 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 17, 2018 |
| Title |
Naive RNA Kaech 1 |
| Sample type |
SRA |
| |
|
| Source name |
Naive RNA Kaech
|
| Organism |
Mus musculus |
| Characteristics |
cell type: CD8+ T cells
cell subset: Naive
assay: RNA
lab: Kaech
run: Sample_N7_006
librarylayout: PAIRED
replicate: 1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
ChIP experiments were performed on FACS purified in vivo adoptively transferred P14+ CD8+ T cells. CD8α+CD44+Thy1.1+ cells were sorted based on expression of KLRG1 and IL7Rα to purified populations of TE (KLRG1HiIL7RαLo) and MP (KLRG1LoIL7RαHi) cells. Cells were crosslinked with 1% formaldehyde in 10% fetal bovine serum containing RPMI medium for 10 minutes at 37C. Crosslinking was stopped by addition of 2.5M glycine at 1:20 dilution for 5 minutes at room temperature. Washed cells were lysed and sonicated to obtain chromatin fragments of 150 to 500 base pairs. ChIP was performed on sonicated chromatin from 1-10 million cells with anti- H3K27me3 (Abcam, ab6002), anti-H3K27Ac (Abcam, ab4729), anti-H3K4me3 (Abcam, ab4XXX), and anti-H3K4me1 (Abcam, ab4XXX) antibodies. ATAC-seq library preparations were performed as described in (Buenrostro et al., 2013). Immunoprecipitated DNA was purified, amplified, processed into a library, and sequenced on an Illumina HiSeq 2500 with 4 samples per lane (170M reads are distributed at 42.5M reads per sample with 75bp reads in single-end mode).
Total RNA was purified with the use of a Qiazol and RNeasy Mini kit (Qiagen), in which on-column treatment with DNase was included. Purified RNA was submitted to the Yale Center for Genomic Analysis, where it was subjected to isolation of mRNA and library preparation. Libraries were pooled, six samples per lane, and were sequenced on an Illumina HISEQ 2500 (75–base pair paired- end reads), followed by alignment and quantification with Kallisto using the GRCm38 (mm10) reference genome. Counts were imported and normalized with DESeq2 v1.14.1, with the read counts normalized by fitting the data to a local smoothed dispersion in an unblinded fit to better capture the observed dispersion-mean relationship and account for batch effects, and subsequently used to perform differential expression analysis (Love et al., 2014).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
N_RNA_Kaech_1
|
| Data processing |
Preprocessing. All samples were subjected to quality control with FastQC. Subsequently, individual reads had adapters removed and required a phred score of 20 or greater to be retained for for further analysis. Reads were mapped using bowtie2 with –very-sensitive settings. Duplicate reads were removed with Picard. In the case of paired-end data, only concordant mate pairs were retained with a mapping score of greater than 30. For ATAC-seq data, mitochondrial reads were excluded and reads were shifted by +4/-5 basepairs on the positive and negative strands, respectively (Langmead & Salzberg, 2012; Buenrostro et al., 2013).
MACS2 Peak Calling. Peaks were first called for each sample using MACS2 v2.1.0, normalizing relative to input in each case. For punctate marks such as H3K27Ac, H3K4me3, and transcription factors, default macs2 settings were used with inputs if available; for broad marks such as H3K27me3, H3K4me1, the –broad setting was invoked with inputs if available; for ATAC-seq, the –broad setting was invoked along with –nomodel, –shift -100, and –extsize 200. (Y. Zhang et al., 2008)
Read counts were first tallied over the atlas peaksets for each mark per sample using HTSeq, ignoring strand and in union mode (Anders et al., 2015). Subsequently, analysis was performed DESeq2 v1.14.1, with the read counts normalized by fitting the data to a local smoothed dispersion unblinded fit to better capture the observed dispersion-mean relationship and account for batch effects (Love et al., 2014).
Genome_build: GRCm38.p5 (mm10)
|
| |
|
| Submission date |
Mar 16, 2018 |
| Last update date |
Mar 21, 2018 |
| Contact name |
Robert Anthony Amezquita |
| E-mail(s) |
robert.a.amezquita@gmail.com
|
| Phone |
8582453350
|
| Organization name |
Fred Hutchinson Cancer Research Institute
|
| Department |
Integrated Immunotherapy Research Center
|
| Lab |
Raphael Gottardo
|
| Street address |
1100 Fairview Ave. N.
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
|
| Relations |
| BioSample |
SAMN08722644 |
| SRA |
SRX3802550 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3045263_N_RNA_Kaech_1_counts.tsv.gz |
1.3 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
