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Sample GSM7906518 Query DataSets for GSM7906518
Status Public on Nov 18, 2023
Title anti-BAF155 CUT&RUN, DN2 and DN3 thymocytes, Bcl11b WT, rep 3
Sample type SRA
 
Source name thymus
Organism Mus musculus
Characteristics tissue: thymus
strain: C57BL/6J
age: 3-6 weeks
Sex: female
cell type: DN23 thymocytes
replicate: 3
genotype: Bcl11bf/f; Vav1-Cre-/-
treatment: NA
Extracted molecule genomic DNA
Extraction protocol CUT&RUN was performed as previously described (Skene et al., eLife, 2017) with minor modifications. Briefly, 500k cells were washed 3x in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x EDTA-free protease inhibitor cocktail (Roche)), then bound to Concanavalin-A beads (Bangs Laboratories) according to manufacturer’s instructions. Cells were incubated with 1:100 dilution of anti-BAF155 antibody (G. Crabtree) overnight at 4 °C in Digitonin Buffer (0.025% digitonin, 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x EDTA-free protease inhibitor cocktail) supplemented with 2 mM EDTA. Sample was then incubated with pA-MNase (S. Henikoff) at 1:200 dilution in Digitonin Buffer at 4 °C for 1h. Digestion was performed under high Ca2+/low salt conditions in Low-Salt Rinse Buffer (3.5 mM HEPES pH 7.5, 0.5 mM spermidine, 0.025% digitonin) at 4 °C for 15 min. The reaction was stopped by the addition of 4x Stop Buffer (final concentration 170 mM NaCl, 10 mM EDTA, 20 mM EGTA, 0.025% digitonin, 25 µg/mL glycogen, 25 µg/mL RNase A (Thermo Fisher)) and the sample was incubated at 37 °C for 30 min. Sample was centrifuged at 16,000g for 10 min at 4 °C, supernatant recovered, and proteins digested in 1x Stop Buffer with 0.1% SDS and 250 µg /mL Proteinase K (New England Biolabs) for 1 hour at 55 °C. CUT&RUN fragments were purified by phenol chloroform extraction.
CUT&RUN libraries were generated using NEBNext UltraII DNA Library Prep Kit for Illumina coupled with NEBNext Multiplex Oligos for Illumina (New England Biolabs) with modifications optimized for small fragments as detailed in dx.doi.org/10.17504/protocols.io.bagaibse.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Data processing Adaptor sequences were trimmed using SeqPurge (v2019_11).
Trimmed reads were mapped to mm10 mouse genome assembly using Bowtie2 (v2.2.9) with settings --local --very-sensitive-local –no-unal –no-mixed –no-discordant –phred33 -I 10 -X 700.
PCR duplicates were removed using Picard (v2.21.8) MarkDuplicates REMOVE_DUPLICATES=true VALIDATION_STRINGENCY=LENIENT.
Reads with MAPQ scores below 30 were purged using samtools (v1.9) view with settings -b -q 30 -f 2 -F 1804.
BedGraph files were generated using bedtools (v2.27.1) genomecov and normalized by the number of fragments intersecting nucleosome-depleted promoter regions (-300 bp to +100 bp) relative to transcriptional start-sites).
BedGraphs were converted to BigWig format using bedGraphToBigWig (v4)
Assembly: mm10
Supplementary files format and content: bigWig
Library strategy: CUT&RUN
 
Submission date Nov 17, 2023
Last update date Nov 18, 2023
Contact name Alexandra Bradu
E-mail(s) abradu@uchicago.edu
Organization name University Of Chicago
Department Pathology
Lab Koh Lab
Street address 924 East 57th Street
City Chicago
State/province IL
ZIP/Postal code 60637
Country USA
 
Platform ID GPL19057
Series (2)
GSE234330 PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape [CUT&RUN]
GSE234331 PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape
Relations
BioSample SAMN38011479
SRA SRX22244298

Supplementary file Size Download File type/resource
GSM7906518_Bcl11b_WT_BAF155-CnR_BioRep3_TechRep1_S7.bw 124.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA

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