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Sample GSM2905798 Query DataSets for GSM2905798
Status Public on Jul 11, 2018
Title Donor1_GFPBATF_antiBATF
Sample type SRA
 
Source name Donor1_wholeblood
Organism Homo sapiens
Characteristics individual: Donor1
cell type: CD4+ T cells
batf tag: GFP-BATF
cut&run antibody: anti-BATF (Santa Cruz, catalog# sc-100974, lot# F2716)
Treatment protocol CUT&RUN was performed using epitope-tagged primary human T cells 11 days after electroporation and 4 days after re-stimulation with anti-CD3/anti-CD28 dynabeads (untagged cells were not electroporated). Approximately 20% and 10% of electroporated cells showed GFP-BATF expression as determined by flow cytometry in donor 1 and donor 2 samples, respectively.
Growth protocol CD4+ T cells were cultured in XVivo15 medium (STEMCELL) with 5% Fetal Bovine Serum, 50 mM 2-mercaptoethanol, and 10 mM N-Acetyl L-Cystine. Immediately following isolation, T cells were stimulated for 2 days with anti-human CD3/CD28 magnetic dynabeads (ThermoFisher) at a beads to cells concentration of 1:1, along with a cytokine cocktail of IL-2 at 200 U/mL (UCSF Pharmacy), IL-7 at 5 ng/mL (ThermoFisher), and IL-15 at 5 ng/mL (Life Tech).  Following electroporation, T cells were cultured in media with IL-2 at 500 U/mL.  Throughout the culture period T cells were maintained at an approximate density of 1 million cells per mL of media.  Every 2-3 days post-electroporation additional media was added, along with additional fresh IL-2 to bring the final concentration to 500 U/mL, and cells were transferred to larger culture vessels as necessary to maintain a density of 1 million cells/mL.
Extracted molecule genomic DNA
Extraction protocol CUT&RUN was performed as described in (https://elifesciences.org/articles/21856), using anti-GFP (ab290), anti-BATF (sc-100974), and rabbit anti-mouse (ab46540) antibodies. Briefly, 6 million cells (30 million cells for anti-GFP CUT&RUN in GFP-BATF-containing cells) were collected and washed. Nuclei were isolated and incubated rotating with primary antibody (GFP or BATF) for 2 hours at 4C. BATF CUT&RUN samples were incubated an additional hour with rabbit anti-mouse antibody. Next, nuclei were incubated with proteinA-micrococcal nuclease (provided by the Henikoff lab) for one hour at 4C. Nuclei were equilibrated to 0C and MNase digestion was allowed to proceed for 30 minutes. Solubilized chromatin CUT&RUN fragments were isolated and purified.
Paired end libraries were generated as described in (https://elifesciences.org/articles/21856). Briefly, purified CUT&RUN DNA underwent end repair followed by ligation to Illumina TruSeq HT dual-indexed adapters. Ligation reactions were followed by two successive Ampure XP bead-based size selections to remove adapter dimers. Libraries were amplified using the KAPA DNA polymerase library preparation kit protocol and amplifying for 14 cycles. Amplified libraries were cleaned up with two successive Ampure XP bead-based size selections to remove adapter dimers.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Data processing We used Bowtie2 2.3.2 with options "--local --very-sensitive-local --no-unal --no-mixed --no-discordant -q --phred33 -I 10 -X 700" to map the paired-end 25bp reads to hg38 of Homo sapiens genomic sequence obtained from UCSC. 
Aligned bam files were converted to bedgraph files and normalized to reads per million using the bedtools v2.26.0 genomecov function function with the "scale" option set to N/10e6, where N = total aligned reads.
Genome_build: UCSC hg38
Supplementary_files_format_and_content: bedgraph
 
Submission date Dec 28, 2017
Last update date Jul 11, 2018
Contact name Ruby Yu
E-mail(s) iamrubyyu@gmail.com
Organization name UCSF
Street address 513 Parnassus Ave
City San Francisco
State/province CA
ZIP/Postal code 94143
Country USA
 
Platform ID GPL18573
Series (1)
GSE108600 CUT&RUN chromatin profiling of endogenously tagged and native BATF
Relations
BioSample SAMN08272651
SRA SRX3518401

Supplementary file Size Download File type/resource
GSM2905798_D1-GFPBATF-aBATF.2.rpm.aligned.bedgraph.gz 571.6 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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