|
| Status |
Public on Feb 11, 2020 |
| Title |
RNA-seq_DP_BL6_Rep1B_Rep2T |
| Sample type |
SRA |
| |
|
| Source name |
CD4+ CD8+ thymocytes
|
| Organism |
Mus musculus |
| Characteristics |
tag: Thymus
tag: C57BL/6J
|
| Treatment protocol |
Thymi of mice were dissociated through a 70µM mesh filter (Falcon) in RPMI 1640 (Corning) with 1% FBS (Sigma-Aldrich), and single cell suspensions were stained with 7AAD (Biolegend) for live cell distinction, as well as PE CD4 (RM404) and APC CD8a (53-6.7) for double-positive CD4+ CD8+ T cell isolation. Sorting was performed on FACS Aria II (BD Biosciences), with forward scatter–height by forward scatter–width and side scatter–height by side scatter–width parameters being used to exclude doublets. Purity was verified after sorting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
washed with 1 x PBS (Corning, cat# 21031CV) and lysed with 350 μl RLT Plus buffer (Qiagen) supplemented with 10% 2-mercaptoethanol (Sigma, cat# M6250), vortexed briefly, snap-frozen on dry ice, and stored at -80°C. Subsequently, total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, cat# 74034).
Libraries were prepared with SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara). Indexed libraries were validated for quality and size distribution using a TapeStation 2200 (Agilent). Paired end sequencing (38 bp+37 bp) was performed on a NextSeq 550 following the manufacturer's protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
RNA-seq libraries were pair-end sequenced by Illumina NextSeq 550. RNA-seq samples were aligned by STAR_2.5.0a_alpha with parameters ‘--readFilesCommand zcat --outFilterMultimapNmax 1--outSAMtype SAM--alignEndsTypeLocal--outReadsUnmapped Fastx--outFilterMismatchNmax 1--alignMatesGapMax 400000--sjdbGTFfile’, with the GTF file being either the mus musculus GRCm38.91 gtf file for C57BL/6 strain or the mus musculus GRCm38.91 gtf shifted to NOD coordinates for the NOD strain.
HTSeq v0.6.1 facilitated counting RNA-seq reads on Gencode vM11 gene models with parameters ‘-s yes-t exon-m intersection-nonempty’.
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text files with read count
|
| |
|
| Submission date |
Dec 11, 2019 |
| Last update date |
Feb 12, 2020 |
| Contact name |
Naomi Goldman |
| Organization name |
University of Pennsylvania
|
| Lab |
Golnaz Vahedi
|
| Street address |
324 BRB II/III, 421 Curie Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE141852 |
Genetic Variations in Type 1 Diabetes Reconfigure the 3D Chromatin Organization of T Cells [RNA-seq] |
| GSE141853 |
Genetic Variations in Type 1 Diabetes Reconfigure the 3D Chromatin Organization of T Cells |
|
| Relations |
| BioSample |
SAMN13541892 |
| SRA |
SRX7348167 |
