 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 21, 2018 |
| Title |
DP scATAC-seq Capture 1 E10 |
| Sample type |
SRA |
| |
|
| Source name |
CD4+CD8+ T cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: CD4+CD8+ T cells (in vivo FACS purified cells)
strain/genotype: C56BL/6 (WT)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA-seq: Total RNA was prepared from approximately 100,000-500,000 cells using an RNeasy Plus Micro Kit (Qiagen). RNA integrity numbers were determined using a TapeStation 2200 (Agilent) and all samples had RIN numbers above 9.5.
ChIP-seq: Cells were chemically cross-linked by 1% formaldehyde. Samples were sonicated and protein-DNA complexes were isolated with antibody.
ATAC-seq: ATAC-seq was performed as previously described with minor modifications (Buenrostro et al., Nature Methods, 2013). Fifty thousand cells from FACS or from cultur were pelleted at 550 x g and washed with 1 mL 1x PBS followed by lysis with 50 μL lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Accessible chromatin was tagmented with Tn5 transposase (FC-121-1030; Illumina) for 45-75 minutes depending on the cell-type in a 37°C waterbath.
scATAC-seq: Single cell ATAC-seq was performed as previously described with minor modifications (Buenrostro et al., Nature, 2015) using the C1 Single-Cell Auto Prep System with the C1 Open App program (Fluidigm). Cells were FACS sorted and stained with mammalian LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen) for 10 minutes on ice at a final concentration of 5 μM Ethidium homodimer-1 and 5 μM Calcein AM in 1x PBS. After staining, cells were diluted in RPMI-1640+1% FBS to a concentration of 400,000 cells per mL. C1 Cell Suspension Reagent (Fluidigm) was added to a final concentration of 20%. Cell capture was verified by light microscope (for Capture 1). Brightfield and fluorescent images of each capture site was taken with a Leica DMi8 (for Captures 2 and 3). The Lysis/Tagmention step in the C1 protocol was lengthened to a duration of 60 minutes using the Open App software (Fluidigm).
RNA-seq: RNA-seq libraries were prepared using the SMARTer High-Input Strand-Specific Total RNA-seq for Illumina kit (Clontech) and were single-end sequenced for 75 bp.
ChIP-seq: ChIP DNA was approximately 300 bp in length. Libraries were prepared using Ultra DNA Library Prep Kit (NEB) and single-end sequenced for 75 bp.
ATAC-seq: Tagmented DNA was amplified for 12 cycles of PCR, purified, and the prepared libraries were paired-end sequenced (38bp+37bp).
scATAC-seq: After single cell ATAC-seq chemistry was performed on the Fluidigm C1, tagmented DNA from single cell libraries were harvested and amplified for 14 PCR cycles. Libraries were pooled and paired-end sequenced (38bp+37bp).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Quality assessment of raw reads was achieved with FastQC and contaminants were removed using Trimgalore with parameters ‘-q 15 --length 20 --stringency 5’. Human (GRCh37, November 17 2015) and mouse (GRCm38, May 23 2014) reference genomes were used in the alignment step. Bulk ATAC-seq samples were mapped to the reference genomes using Bowtie2 v2.2.9 with ‘-X2000’. STAR v2.5 was used for aligning single-cell ATAC-, RNA-, ChIP- and MNase-seq reads with parameters specifically optimized for each protocol. RNA-seq samples were analyzed with parameters ‘--outFilterMultimapNmax 1 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --alignEndsType Local’. ChIP-seq raw reads were aligned with parameters ‘--alignSJDBoverhangMin 999 --alignSJoverhangMin 999 --alignIntronMax 1 --outFilterMultimapNmax 1 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --alignEndsType Local’ to disable the usage of known and prevent calling novel splice junctions. The same parameters were also applied for mapping MNase-seq data combined with ‘--alignMatesGapMax 2000’. Reads aligned to the mitochondrial genome as well as reads mapping to multiple genomic loci were discarded. Picard minimized the PCR amplification bias in ATAC-, ChIP- and MNase-seq samples. In paired-end MNase-seq samples, fragments smaller than 75bp were filtered out. For calling peaks in bulk ATAC-seq, macs2 with parameters ‘-p 1e-7 --nolambda --nomodel’ was used. Peak calling on merged single-cell ATAC-seq samples was facilitated with macs2 using parameters ‘-p 1e-3 --nolambda --nomodel’. Peaks for transcription factor ChIP-seq samples were called using macs2 with parameters ‘-p 1e-3 -q 0.05’. For calling reproducible peaks in Tcf-1 ChIP-seq samples from NIH3T3 Tcf-1 RV cells, macs2 was initially applied on each of the two replicates as well as after merging both replicates with parameters ‘--nomodel --extsize 300 --keep-dup all --call-summits -q 0.9’ using the Tcf-1 ChIP-seq on NIH3T3 Empty RV cells as control. The identified peaks were filtered with Irreproducible Discovery Rate (IDR) v2.0.2 using an IDR threshold of 2e-2.
Genome_build: mm10
|
| |
|
| Submission date |
May 22, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Herbert R De'Broski |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Pennsylvania
|
| Street address |
3800 Spruce St
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL21626 |
| Series (1) |
| GSE99159 |
Tcf-1 shapes the chromatin landscape of naive T cells |
|
| Relations |
| BioSample |
SAMN07151724 |
| SRA |
SRX2840601 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2634418_scATAC-seq_DP_WT_E10_21822846_S58.bw |
48.6 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
