Grace’s Notebook: Samples in Lyophilizer got all over the place

At 10:30 am, I went to the lyoppilizer room and met up with Megan. My samples had gotten all over the inside of the manifold. Sam thinks it’s likely because the sampels thawed before they got on the lyophilizer, which is very likely because I brought them over on wet ice as opposed to dry ice. WIll try again at 4:30pm (bringing them over on dry ice) and Sam will grab them in the morning on Thursday. I will also test this out only with screw cap tubes and have them slightly unscrewed. Megan also noticed that the vacuum didn’t get below 100 mtorr, which it should… so the manifold likely wasn’t sealed properly. So Megan added a lot more grease.

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Yaamini’s Notebook: DML Analysis Part 7

DML-mRNA Gene Enrichment

My revised blastx finished running over the weekend! The output can be found here. I emailed the revised blast output and original transcript file to Mike Riffle in Genome Sciences so he could modify the gene enrichment tool.

DML-mRNA Gene Enrichment

In my R Markdown file, I merged the transcript-uniprot blast results with the DML-mRNA overlap file. I exported the results in this file.

I isolated the Uniprot accession codes so I could do a standard DAVID gene enrichment. I unfolded columns in both the transcript blastx results and DML-mRNA overlaps and saved them as new files. I also saved the Uniprot codes for the background (transcripts) and gene list (overlaps) in this document.

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I saved the DAVID output in this folder. I downloaded information for all functional annotations, as well as individual lists for biological processes, cellular components, and molecular functions.

I wanted to use ReVIGO to visualize significant GOterms, but ReVIGO’s interactive graphic wasn’t loading after I pasted a list of GOterms and p-values! I posted this issue. I have the option to download an R script to create the graphic, which would be useful, but also extremely tedious for image formatting. If I don’t hear anything by tomorrow morning, I’ll need to download the R scripts. My goal is to present the DML-mRNA enrichment results at PCSGA.

DML-Exon Gene Enrichment

Steven confirmed I need to do a gene enrichment for DML-exon, DML-intron, flanking regions, and all regions combined. I thought I could get started on these enrichments, even though I’ll probably concentrate on the DML-mRNA enrichment for my PCSGA talk. However, the DML-exon file doesn’t have the same transcript IDs the DML-mRNA and blastx results do. The DML-exon and DML-intron files only have chromosome IDs, positions, and strand information. I posted another issue asking if it made sense to merge the DML-exon or DML-intron files with the original mRNA gff to obtain transcript IDs. If it doesn’t, I’ll need to figure out another way to do that gene enrichment.

Going forward

While I wait for the verdict on my two open issues, I’m going to start drafting my PCSGA talk, focusing on the DML-mRNA enrichment. I saw lots of significant GOterms related to reproduction and growth, so that will be interesting to explain!

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Grace’s Notebook: Start Lyophilizing to test new RNA extraction protocol using Tri-reagent

Today I started using the lyophilizer on the samples I picked out (pellet and supernatant) and the pooled sample of the ones from Sam’s Qiagen RNeasy extraction that had “Out of range” Qubit results.

Lyophilizer

Start: 10:45am 09/10/18
Samples:
pooled sample of the ones from Sam’s Qiagen RNEasy extraction that had “out of range” Qubit results
AND
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Lyophilizer setup:
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In order to use (there are instructions on how to use that are posted in the room):

  • make sure that the condenser (inside the machine- visible when you remove the cylindrical container and the cover) is dry
  • turn on condenser
  • wipe off all the old grease on the rim of the opening to the condenser and the black tube
  • Add new grease to the rim and the black tube
  • place the cover, the rack with samples on it, and the cylindrical container with the black rubber tube along the bottom
  • make sure all of the valves on openings at the top of the cylindrical container are vertical (lined up with the black notch)
  • Once the condenser gets below -70 C, turn on the vacuum

Megan Feddern (Holtgrieve Lab) helped me with all of this today and tomorrow she’ll show me how to take out my samples. She is sharing the lyophilizer with me.

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