Posts - Page 2 of 148
Data Wrangling - C.virginica Genes Only FastA from Genes BED File Using gffread on Raven
I’ve been reviewing some of the CEABIGR (GitHub repo) data I’ve generated; specifically transcript count data/calcs. As part of that, I feel like we need/should annotate the transcripts to be able to make some more informed conclusions. Steven had previously performed annotations (Notebook), as well as Yaamini (GitHub Issue). However, there are shortcomings to both of the approaches each one utilized. Steven’s annotation relied only on coding sequences (CDS), while Yaamini’s utilized only mRNAs.
Read Extractions - M.magister MEGAN Arthropoda and Unassigned Reads to FastA
After converting DIAMOND BLASTx/MEGAN DAA files to RMA6 and looking at the taxonomic breakdown of each sample (notebook entry), it was decided we should extract reads from Arthropoda (and all taxonomies below) and all Unassigned reads from each sample. To do so, I used MEGAN6 Community Edition.
RNA Quantification - C.gigas PolyIC
Steven asked me to generate cDNA (GitHub Issue) from Crassostrea gigas (Pacific oyster) RNA, as part of Matt George’s polyIC project. Before starting reverse transcription, I felt it would be best to quantify the RNA using the Qubit 3.0 using the Broad Range Assay. Steven and I discussed which samples to use earlier this week.
Reverse Transcription - C.gigas PolyIC RNA
Quantified RNA earlier today and proceeded to make cDNA. Reverse transcription was performed using oligo dT primers using M-MLV RT (Promega), per the manufacturer’s recommendations. Used 400ng of RNA in each reaction. I used 400ng (instead of the usual 100ng) to simplify pipetting for high concentration samples, without the need/time to dilute samples. All reactions were done on ice in 0.5uL PCR tubes.
qPCR - C.gigas Ctenidia cDNA for Noah’s Heat-Mechanical Stress Project
Using cDNA from 20230713, performed qPCR on the following primer sets selected by Steven:
qPCR - C.gigas Ctenidia cDNA for Noah’s Heat-Mechanical Stress Project
Using cDNA from 20230713, performed qPCR on the following primer sets selected by Steven:
qPCR - C.gigas cDNA Primer Tests for Noah’s Heat-Mechanical Stress Project
After generating cDNA earlier today from Noah’s Crassostrea gigas (Pacific oyster) RNA (isolated 20230712), I ran qPCRs using the pooled cDNA sample to test out some primers that Steven pulled from the freezer.
Reverse Transcription - C.gigas RNA from Noah’s Heat-Mechanical Stress Project
After isolating ctenidia RNA on 20230712 and then quantifying the RNA earlier today, I needed to perform reverse transcription to have cDNA for subsequent qPCRs. Reverse transcription was performed using oligo dT primers using M-MLV RT (Promega), per the manufacturer’s recommendations. Used 100ng of RNA in each reaction. All reactions were done on ice in 0.5uL PCR tubes.
RNA Quantification - C.gigas RNA from Noah’s Heat-Mechanical Stress Project
I quantified Noah’s Crassostrea gigas (Pacific oyster) ctenidia RNA from yesterday (20230712) using the Roberts Lab Qubit 3 and the RNA High Sensitivity (HS) Assay. 1uL of sample was used for each measurement.
RNA Isolation - C.gigas Ctenidia from Noah’s Heat-Mechanical Stress Project
Steven asked that I help isolate RNA (GitHub Issue) for Noah’s summer project involving heat and mechanical stress (tumbling) of adult Crassostrea gigas (Pacific oyster). Here’s a brief overview of the process: