|
| Status |
Public on Nov 15, 2017 |
| Title |
Sample 14 DHFR.YFP DMSO 24h |
| Sample type |
SRA |
| |
|
| Source name |
DHFR.YFP DMSO 24h
|
| Organism |
Canis lupus familiaris |
| Characteristics |
cell type: Madin-Darby Canine Kidney (MDCK) cells
genotype/variation: Clonal; stably expressing DHFR.YFP
developmental stage: Adult-derived epithelial cell line
treated with: 0.1% DMSO for 24hrs
|
| Treatment protocol |
At the time of plating, cells were treated with either 0.1% DMSO, 10 µM TMP, or 10 nM STA-9090 (24 hour treatment).
|
| Growth protocol |
Low passage cells were plated at a density of 200,000 cells/well in 6 well plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were trypsinized and RNA was extracted using the Qiagen RNeasy Plus Mini Kit and QIAshredder homogenization columns.
For RNAseq, RNA integrity and concentration were checked on a Fragment Analyzer (Advanced Analytical) and libraries were prepared using the Illumina NeoPrep RNAseq kit. Libraries were quantified using the Fragment Analyzer (Advanced Analytical) and qPCR before being loaded for single-end sequencing using the Illumina HiSeq 2000 for 50 nucleotides.
polyA-based mRNA capture using Neoprep with TruSeq strand-specific strategy.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
D17-3259
|
| Data processing |
Illumina Offline BaseCaller1.9.3 software used for basecalling.
Quality control: Reads were aligned against canFam3 (Sept. 2011) using bwa mem v. 0.7.12-r1039 with flags –t 16 –f and mapping rates, fraction of multiply-mapping reads, number of unique 20-mers at the 5’ end of the reads, insert size distributions and fraction of ribosomal RNAs were calculated using dedicated perl scripts and bedtools v. 2.25.0.64 In addition, each resulting bam file was randomly down-sampled to a million reads, which were aligned against canFam3 and read density across genomic features were estimated for RNA-Seq-specific quality control metrics.
RNA-Seq mapping and quantitation: Reads were aligned against canFam3 / ENSEMBL 86 annotation using STAR v. 2.5.3a with flags -runThreadN 8 --runMode alignReads --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 10 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM with --genomeDir pointing to a 75nt-junction canFam3 STAR suffix array.
Gene expression was quantitated using RSEM v. 1.3.0 with the following flags for all libraries: rsem-calculate-expression --calc-pme --alignments -p 8 --forward-prob 0 against an annotation matching the STAR SA reference. Posterior mean estimates (pme) of counts and estimated RPKM were retrieved.
Genome_build: canFam3
Supplementary_files_format_and_content: Tab-delimited text file containing log2-transformed RPKM values for all canFam3 annotated genes (ENSEMBL 86) in each sample (RSEM pme_RPKM output).
|
| |
|
| Submission date |
Aug 18, 2017 |
| Last update date |
Dec 19, 2023 |
| Contact name |
Matthew D Shoulders |
| E-mail(s) |
mshoulde@mit.edu
|
| Organization name |
Massachusetts Institute of Technology
|
| Department |
Chemistry
|
| Street address |
77 Massachusetts Avenue
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02446 |
| Country |
USA |
| |
|
| Platform ID |
GPL16540 |
| Series (1) |
| GSE102797 |
Host Proteostasis Modulates Influenza Evolution |
|
| Relations |
| BioSample |
SAMN07516122 |
| SRA |
SRX3102649 |
