Yaamini’s Notebook: Gigas Labwork Plans

Gearing up for C. gigas labwork

Now that my C. virginica gonad methylation paper is in review (crosses fingers), I’m ready to tackle the next phase of sample preparation and sequencing. I already processed pooled C. gigas gonad DNA samples for WGBS and completed a preliminary analysis of that data. I still have a good amount of gonad DNA that can be processed for bisulfite sequencing, remaining tissue on histology blocks that can be used for RNA-Seq, other broodstock tissues from the same oysters in the freezer, and even some larvae. After chatting with Steven, we outlined 2.5ish projects for me to work on:

1. Potential for intergenerational epigenetic inheritance in C. gigas larvae

After counting larvae 18 hours post-fertilization and stocking larval rearing buckets at the appropriate density, I had some larvae left over from each family:

  • ambient pH females (pool) x ambient pH males
  • ambient pH females (pool) x low pH males
  • low pH females (pool) x ambient pH males
  • low pH females (pool) x low pH males

With only four samples total I don’t have a very robust experiment, but it should be very easy for me to extract DNA for MBD-Seq and RNA and compare patterns in larvae with the adult gonad samples.

2. Examination of various epigenetic and genetic mechanisms in response to ocean acidification

I’m interested in seeing how epigenetic and genetic mechanisms play together to influence responses to ocean acidification! I have remaining gonad tissue in histology blocks and frozen adductor, ctenidia, and mantle tissues from C. gigas broodstock in low pH (10 individuals) and ambient pH (also 10 individuals). I have more than enough frozen tissue for DNA and RNA extractions, ATAC-Seq, and maybe even proteomics.

Since some gonad DNA has already been used for WGBS libraries, I will look into using low volume MBD-BSSeq for the remaining gonad DNA. I’ve already looked into low-input Qiagen kits (and I think it’s discontinued?), but using a modified protocol of the MethylMiner kit that I’ve already used before and know works seems like a safer option.

I’ll extract RNA from the remaining tissue in the histology blocks, which is something Laura did previously. I’ll look at her protocol and try it out on a few samples. Extracting DNA and RNA from frozen somatic tissues shouldn’t be any problem at all! There may be a kit I could use to extract both DNA for WGBS and RNA for RNA-Seq from the tissue, but I also have more than enough tissue to do separate extractions. I’ll also save some tissue for proteomics if we decide to go down that route later. My goal is to prepare as many samples as I can and worry about sequencing decisions later.

Now the complicated part: ATAC-Seq. I’d like to replicate the analysis in the Gatzmann et al. 2018 paper to link gene expression with gene accesibility. It seems like every ATAC-Seq protocol requires single-cell suspensions, so scATAC-Seq may be the only option available. During Science Hour, Shelly emailed the individual that developed scATAC-Seq and graduate student working on ATAC-Seq in frozen salmon tissue to see if they had any suggestions about doing ATAC-Seq with frozen mollusc tissue.

2.5 Ocean acidification impacts on fertilization with ATAC-Seq

While talking about ways to get single cells from frozen tissues or gonad histology blocks, Steven realized that Mac is fertilizing C. giags gametes and isolating single cells from embryos and larvae every other Wednesday for scRNA-Seq. If I were able to fertilize some gametes in low pH conditions and others in ambient pH conditions, I could isolate single cells from these larvae. Since the scRNA-Seq could be paid for on another grant, I could do complimentary scATAC-Seq. I will shadow Mac next Wednesday to see what she’s doing and figure out if I could use similar techniques.

Going forward

  1. Shadow Mac for single cell experiments
  2. Determine what kits to use and order necessary materials
  3. Test RNA extraction protocol with tissue in histology blocks
  4. Start processing frozen tissues
  5. Extract DNA and RNA from larvae
  6. Identify an ATAC-Seq protocol and start testing it
  7. Figure out what to do with C. virginica sperm and other potential samples

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Sam’s Notebook: RNA Isolation and Quantification – C.bairdi Hemocyte Pellets in RNAlater

Isolated RNA from the following hemolymph pellet samples:

  • 6202_337_12
  • 6142_474_26
  • 6144_477_26
  • 6122_489_26
  • 6176_490_26
  • 6136_491_26
  • 6137_500_26
  • 6125_503_26
  • 6145_505_26
  • 6131_507_26
  • 6159_508_26
  • 6206_509_26
  • 6150_511_26
  • 6161_512_26
  • 6115_514_26
  • 6179_515_26
  • 6172_517_26

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • Performed on-column DNase step.
  • RNA was eluted in 15uL H2O

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.

RNA was stored in the [-80o</sup>C freezer](http://b.link/srlab-80C) in Shellfish Box #8.

Sam’s Notebook: RNA Isolation and Quantification – C.bairdi Hemocyte Pellets in RNAlater

Isolated RNA from the following hemolymph pellet samples:

  • 6174_233_12
  • 6258_261_12
  • 6270_269_12
  • 6161_302_12
  • 6115_307_12
  • 6206_319_12
  • 6241_323_12
  • 6158_324_12
  • 6272_338_12
  • 6152_344_12
  • 6275_372_12
  • 6218_373_12
  • 6153_404_26
  • 6132_406_26
  • 6162_407_26
  • 6178_410_26
  • 6163_424_26
  • 6140_429_26
  • 6174_433_26
  • 6199_445_26
  • 6156_446_26
  • 6106_450_26
  • 6151_454_26
  • 6177_456_26
  • 6119_461_26
  • 6124_464_26
  • 6134_465_26
  • 6168_466_26
  • 6152_467_26
  • 6121_472_26
  • 6205_475_26
  • 6155_476_26
  • 6143_479_26
  • 6158_483_26
  • 6169_486_26

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • Performed on-column DNase step.
  • RNA was eluted in 15uL H2O

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.

RNA was stored in the [-80o</sup>C freezer](http://b.link/srlab-80C) in Shellfish RNA Box #7 and Shellfish Box #8.

Sam’s Notebook: Transdecoder – Hematodinium MEGAN6 Taxonomic-Specific Reads Assembly from 20200122

Ran Trinity to de novo assembly on the the C.bairdi MEGAN6 taxonomic-specific RNAseq data on 201200122 and now will begin annotating the transcriptome using TransDecoder on Mox.

SBATCH script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=transdecoder_hemat ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200123_hemat_transdecoder_megan # Exit script if a command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # Set workind directory as current directory wd="$(pwd)" # Capture date as YYYYMMDD timestamp=$(date +%Y%m%d) # Set input file locations and species designation trinity_fasta="/gscratch/srlab/sam/data/Hematodinium/transcriptomes/20200122.hemat.megan.Trinity.fasta" trinity_gene_map="/gscratch/srlab/sam/data/Hematodinium/transcriptomes/20200122.hemat.megan.Trinity.fasta.gene_trans_map" species="hemat" # Capture trinity file name trinity_fasta_name=${trinity_fasta##*/} # Paths to input/output files blastp_out_dir="${wd}/blastp_out" transdecoder_out_dir="${wd}/${trinity_fasta_name}.transdecoder_dir" pfam_out_dir="${wd}/pfam_out" blastp_out="${blastp_out_dir}/${timestamp}.${species}.blastp.outfmt6" pfam_out="${pfam_out_dir}/${timestamp}.${species}.pfam.domtblout" lORFs_pep="${transdecoder_out_dir}/longest_orfs.pep" pfam_db="/gscratch/srlab/programs/Trinotate-v3.1.1/admin/Pfam-A.hmm" sp_db="/gscratch/srlab/programs/Trinotate-v3.1.1/admin/uniprot_sprot.pep" # Paths to programs blast_dir="/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin" blastp="${blast_dir}/blastp" hmmer_dir="/gscratch/srlab/programs/hmmer-3.2.1/src" hmmscan="${hmmer_dir}/hmmscan" transdecoder_dir="/gscratch/srlab/programs/TransDecoder-v5.5.0" transdecoder_lORFs="${transdecoder_dir}/TransDecoder.LongOrfs" transdecoder_predict="${transdecoder_dir}/TransDecoder.Predict" # Make output directories mkdir "${blastp_out_dir}" mkdir "${pfam_out_dir}" # Extract long open reading frames "${transdecoder_lORFs}" \ --gene_trans_map "${trinity_gene_map}" \ -t "${trinity_fasta}" # Run blastp on long ORFs "${blastp}" \ -query "${lORFs_pep}" \ -db "${sp_db}" \ -max_target_seqs 1 \ -outfmt 6 \ -evalue 1e-5 \ -num_threads 28 \ > "${blastp_out}" # Run pfam search "${hmmscan}" \ --cpu 28 \ --domtblout "${pfam_out}" \ "${pfam_db}" \ "${lORFs_pep}" # Run Transdecoder with blastp and Pfam results "${transdecoder_predict}" \ -t "${trinity_fasta}" \ --retain_pfam_hits "${pfam_out}" \ --retain_blastp_hits "${blastp_out}" 

Sam’s Notebook: Transdecoder – C.bairdi MEGAN6 Taxonomic-Specific Reads Assembly from 20200122

Ran Trinity to de novo assembly on the the Hematodinium MEGAN6 taxonomic-specific RNAseq data on 201200122 and now will begin annotating the transcriptome using TransDecoder on Mox.

SBATCH script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=transdecoder_cbai ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200123_cbai_transdecoder_megan # Exit script if a command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # Set workind directory as current directory wd="$(pwd)" # Capture date as YYYYMMDD timestamp=$(date +%Y%m%d) # Set input file locations and species designation trinity_fasta="/gscratch/srlab/sam/data/C_bairdi/transcriptomes/20200122.C_bairdi.megan.Trinity.fasta" trinity_gene_map="/gscratch/srlab/sam/data/C_bairdi/transcriptomes/20200122.C_bairdi.megan.Trinity.fasta.gene_trans_map" species="cbai" # Capture trinity file name trinity_fasta_name=${trinity_fasta##*/} # Paths to input/output files blastp_out_dir="${wd}/blastp_out" transdecoder_out_dir="${wd}/${trinity_fasta_name}.transdecoder_dir" pfam_out_dir="${wd}/pfam_out" blastp_out="${blastp_out_dir}/${timestamp}.${species}.blastp.outfmt6" pfam_out="${pfam_out_dir}/${timestamp}.${species}.pfam.domtblout" lORFs_pep="${transdecoder_out_dir}/longest_orfs.pep" pfam_db="/gscratch/srlab/programs/Trinotate-v3.1.1/admin/Pfam-A.hmm" sp_db="/gscratch/srlab/programs/Trinotate-v3.1.1/admin/uniprot_sprot.pep" # Paths to programs blast_dir="/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin" blastp="${blast_dir}/blastp" hmmer_dir="/gscratch/srlab/programs/hmmer-3.2.1/src" hmmscan="${hmmer_dir}/hmmscan" transdecoder_dir="/gscratch/srlab/programs/TransDecoder-v5.5.0" transdecoder_lORFs="${transdecoder_dir}/TransDecoder.LongOrfs" transdecoder_predict="${transdecoder_dir}/TransDecoder.Predict" # Make output directories mkdir "${blastp_out_dir}" mkdir "${pfam_out_dir}" # Extract long open reading frames "${transdecoder_lORFs}" \ --gene_trans_map "${trinity_gene_map}" \ -t "${trinity_fasta}" # Run blastp on long ORFs "${blastp}" \ -query "${lORFs_pep}" \ -db "${sp_db}" \ -max_target_seqs 1 \ -outfmt 6 \ -evalue 1e-5 \ -num_threads 28 \ > "${blastp_out}" # Run pfam search "${hmmscan}" \ --cpu 28 \ --domtblout "${pfam_out}" \ "${pfam_db}" \ "${lORFs_pep}" # Run Transdecoder with blastp and Pfam results "${transdecoder_predict}" \ -t "${trinity_fasta}" \ --retain_pfam_hits "${pfam_out}" \ --retain_blastp_hits "${blastp_out}" 

Sam’s Notebook: Transcriptome Annotation – Hematodinium MEGAN Trinity Assembly Using DIAMOND BLASTx on Mox

As part of annotating the transcriptome assembly from the MEGAN6 Hematodinium taxonomic-specific reads, I need to run DIAMOND BLASTx to use with Trinotate.

Ran DIAMOND BLASTx against the UniProt/SwissProt database (downloaded today) on Mox.

SBATCH script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=hemat_blastx_DIAMOND ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200123_hemat_diamond_blastx_megan # Exit script if any command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # SegFault fix? export THREADS_DAEMON_MODEL=1 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # Program paths diamond=/gscratch/srlab/programs/diamond-0.9.29/diamond # DIAMOND UniProt database dmnd=/gscratch/srlab/blastdbs/uniprot_sprot_20200123/uniprot_sprot.dmnd # Trinity assembly (FastA) fasta=/gscratch/srlab/sam/data/Hematodinium/transcriptomes/20200122.hemat.megan.Trinity.fasta # Strip leading path and extensions no_path=$(echo "${fasta##*/}") no_ext=$(echo "${no_path%.*}") # Run DIAMOND with blastx # Output format 6 produces a standard BLAST tab-delimited file ${diamond} blastx \ --db ${dmnd} \ --query "${fasta}" \ --out "${no_ext}".blastx.outfmt6 \ --outfmt 6 \ --evalue 1e-4 \ --max-target-seqs 1 \ --block-size 15.0 \ --index-chunks 4 

Sam’s Notebook: Transcriptome Annotation – C.bairdi MEGAN Trinity Assembly Using DIAMOND BLASTx on Mox

As part of annotating the transcriptome assembly from the MEGAN6 C.bairdi taxonomic-specific reads, I need to run DIAMOND BLASTx to use with Trinotate.

Ran DIAMOND BLASTx against the UniProt/SwissProt database (downloaded today) on Mox.

SBATCH script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=cbai_blastx_DIAMOND ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200123_cbai_diamond_blastx_megan # Exit script if any command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # SegFault fix? export THREADS_DAEMON_MODEL=1 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # Program paths diamond=/gscratch/srlab/programs/diamond-0.9.29/diamond # DIAMOND UniProt database dmnd=/gscratch/srlab/blastdbs/uniprot_sprot_20200123/uniprot_sprot.dmnd # Trinity assembly (FastA) fasta=/gscratch/srlab/sam/data/C_bairdi/transcriptomes/20200122.C_bairdi.megan.Trinity.fasta # Strip leading path and extensions no_path=$(echo "${fasta##*/}") no_ext=$(echo "${no_path%.*}") # Run DIAMOND with blastx # Output format 6 produces a standard BLAST tab-delimited file ${diamond} blastx \ --db ${dmnd} \ --query "${fasta}" \ --out "${no_ext}".blastx.outfmt6 \ --outfmt 6 \ --evalue 1e-4 \ --max-target-seqs 1 \ --block-size 15.0 \ --index-chunks 4 

Sam’s Notebook: Transcriptome Assembly – Hematodinium with MEGAN6 Taxonomy-specific Reads with Trinity on Mox

Ran a de novo assembly using [the extracted reads classified under Alveolata from 20200122].(https://ift.tt/2RhkjuM) The assembly was performed with Trinity on Mox.

SBATCH script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=trinity_hemat ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200122_hemat_trinity_megan_RNAseq # Exit script if a command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # User-defined variables reads_dir=/gscratch/srlab/sam/data/C_bairdi/RNAseq threads=27 assembly_stats=assembly_stats.txt timestamp=$(date +%Y%m%d) fasta_name="${timestamp}.hemat.megan.Trinity.fasta" # Paths to programs trinity_dir="/gscratch/srlab/programs/trinityrnaseq-v2.9.0" samtools="/gscratch/srlab/programs/samtools-1.10/samtools" ## Inititalize arrays R1_array=() R2_array=() # Variables for R1/R2 lists R1_list="" R2_list="" # Create array of fastq R1 files R1_array=(${reads_dir}/*_R1.fq) # Create array of fastq R2 files R2_array=(${reads_dir}/*_R2.fq) # Create list of fastq files used in analysis ## Uses parameter substitution to strip leading path from filename for fastq in ${reads_dir}/*.fq do echo "${fastq##*/}" >> fastq.list.txt done # Create comma-separated lists of FastQ reads R1_list=$(echo "${R1_array[@]}" | tr " " ",") R2_list=$(echo "${R2_array[@]}" | tr " " ",") # Run Trinity using "stranded" setting (--SS_lib_type) ${trinity_dir}/Trinity \ --seqType fq \ --max_memory 120G \ --CPU ${threads} \ --SS_lib_type RF \ --left "${R1_list}" \ --right "${R2_list}" # Rename generic assembly FastA mv trinity_out_dir/Trinity.fasta trinity_out_dir/${fasta_name} # Assembly stats ${trinity_dir}/util/TrinityStats.pl trinity_out_dir/${fasta_name} \ > ${assembly_stats} # Create gene map files ${trinity_dir}/util/support_scripts/get_Trinity_gene_to_trans_map.pl \ trinity_out_dir/${fasta_name} \ > trinity_out_dir/${fasta_name}.gene_trans_map # Create FastA index ${samtools} faidx \ trinity_out_dir/${fasta_name} 

Sam’s Notebook: Transcriptome Assembly – C.bairdi with MEGAN6 Taxonomy-specific Reads with Trinity on Mox

Ran a de novo assembly using [the extracted reads classified under Arthropoda from 20200122].(https://ift.tt/2RhkjuM) The assembly was performed with Trinity on Mox.

SBATCH Script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=trinity_cbai ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=500G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200122_cbai_trinity_megan_RNAseq # Exit script if a command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # User-defined variables reads_dir=/gscratch/srlab/sam/data/C_bairdi/RNAseq threads=27 assembly_stats=assembly_stats.txt timestamp=$(date +%Y%m%d) fasta_name="${timestamp}.C_bairdi.megan.Trinity.fasta" # Paths to programs trinity_dir="/gscratch/srlab/programs/trinityrnaseq-v2.9.0" samtools="/gscratch/srlab/programs/samtools-1.10/samtools" ## Inititalize arrays R1_array=() R2_array=() # Variables for R1/R2 lists R1_list="" R2_list="" # Create array of fastq R1 files R1_array=(${reads_dir}/*_R1.fq) # Create array of fastq R2 files R2_array=(${reads_dir}/*_R2.fq) # Create list of fastq files used in analysis ## Uses parameter substitution to strip leading path from filename for fastq in ${reads_dir}/*.fq do echo "${fastq##*/}" >> fastq.list.txt done # Create comma-separated lists of FastQ reads R1_list=$(echo "${R1_array[@]}" | tr " " ",") R2_list=$(echo "${R2_array[@]}" | tr " " ",") # Run Trinity using "stranded" setting (--SS_lib_type) ${trinity_dir}/Trinity \ --seqType fq \ --max_memory 500G \ --CPU ${threads} \ --SS_lib_type RF \ --left "${R1_list}" \ --right "${R2_list}" # Rename generic assembly FastA mv trinity_out_dir/Trinity.fasta trinity_out_dir/${fasta_name} # Assembly stats ${trinity_dir}/util/TrinityStats.pl trinity_out_dir/${fasta_name} \ > ${assembly_stats} # Create gene map files ${trinity_dir}/util/support_scripts/get_Trinity_gene_to_trans_map.pl \ trinity_out_dir/${fasta_name} \ > trinity_out_dir/${fasta_name}.gene_trans_map # Create FastA index ${samtools} faidx \ trinity_out_dir/${fasta_name} 

Sam’s Notebook: RNA Isolation and Quantification – C.bairdi Hemocyte Pellets in RNAlater Troubleshooting

After the failure to obtain RNA from any C.bairdi hemocytes pellets (out of 24 samples processed) on 20200117, I decided to isolate RNA from just a subset of that group to determine if I screwed something up last time or something. Also, I am testing two different preparations of the kit-supplied DNase I: one Kaitlyn prepped and a fresh preparation that I made. Admittedly, I’m not doing the “proper” testing by trying the different DNase preps on the same exact sample, but it’ll do. I just want to see if I get some RNA from these samples this time…

Isolated RNA from the following 4 hemolymph pellet samples:

  • 6128_112_9 (Kaitlyn DNase)
  • 6204_114_9 (Kaitlyn DNase)
  • 6141_123_9 (Sam DNase)
  • 6245_126_9 (Sam DNase)

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • Performed on-column DNase step.
  • RNA was eluted in 15uL H2O

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.