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| Status |
Public on Feb 15, 2024 |
| Title |
S4_Input_UN_FLAGx3BAP1 |
| Sample type |
SRA |
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| Source name |
CH12F3
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| Organism |
Mus musculus |
| Characteristics |
cell line: CH12F3
cell type: B lymphocytes
chip antibody: n/a
treatment: unstimulated
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CH12F3 cells were either unstimulated or stimulated with a cocktail consisting of recombinant human TGFβ1 (R&D systems, 240-B, 1 ng/mL), recombinant mouse IL4 (Preprotech, 214-14, 10 ng/mL), and functional grade purified α-mouse CD40 (eBiosciences, 16-0401, 1 ug/mL) for 72 hours to induce IgA class-switching.
Cells were fixed with 1% formaldehyde in the culture media for 10 minutes at room temperature, followed by addition of 0.125M of glycine to stop fixation. Nuclei were extracted with 5 minutes lysis in 0.25% Triton buffer, followed by 30 minutes in 200mM NaCl buffer. Nuclei were resuspended in sonication buffer and sonicated for twelve cycles of 30 seconds with a digital sonifier (Branson Ultrasonics) at 80%, with 30 seconds rest in cooled circulating water. Beads immunocomplexes were prepared overnight by conjugating 40μL of Dynabeads Protein G (Invitrogen, Life Technologies) with antibodies: anti-BAP1 (Cell Signaling Technology, D1W9B, 52.8 μg), anti-FLAG M2 (Sigma, F1804, 5 μg) or anti-H2AK119Ub (Cell Signaling Technology, D27C4, 5 μg). Immunoprecipitation was performed by overnight incubation of antibody-bead matrices with sheared chromatin from the equivalent of 5x106 cells. For BAP1-FLAG ChIP, 6 washes were performed with medium-stringency buffers, while 4 low-stringency washes were used for histone ChIP . Samples were de-crosslinked by overnight incubation at 65̊C in 1% SDS buffer. Following RNaseA and Proteinase K enzymatic treatments, ChIP DNA was purified using Qiaquick PCR Cleanup kit (Qiagen).
DNA yields and quality were assessed using Bioanalyzer (Agilent). Library preparation were performed using the KAPA HyperPrep Kit. Sequencing was assessed using Novaseq 6000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The quality of the sequencing reads was confirmed using the FastQC tool (Babraham Bioinformatics), and low-quality bases were trimmed from the read extremities using Trimmomatic v.0.33. The reads were mapped to the UCSC mouse mm9 reference genome with Bowtie 1.0.0, and BAP1 binding sites identified using peak detection algorithm MACS1.4, with comparisons for read enrichment against control input DNA from the same cells. Normalized sequence read density profiles (bigwig) were generated with Homer tool and visualized with IGV.
Gene ontology (GO) and disease ontology enrichment analyses on genes associated with BAP1 ChIP-Seq binding clusters were performed on GREAT 4.0.4 with Basal plus extension option, searching for genes within 2kb upstream, 2kb downstream, and 200kb in distal.
Assembly: mm9
Supplementary files format and content: bigwig files
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| Submission date |
Oct 12, 2023 |
| Last update date |
Feb 15, 2024 |
| Contact name |
Anastasia Nijnik |
| E-mail(s) |
anastasia.nijnik@mcgill.ca
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| Phone |
514-398-5567
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| Organization name |
McGill University
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| Department |
Physiology
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| Lab |
Nijnik Lab
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| Street address |
Rm 368 - 3649 Promenade Sir William Osler
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G 0B1 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE245220 |
B-cell Intrinsic Regulation of Antibody Mediated Immunity by Histone H2A Deubiquitinase BAP1 [ChIP-seq] |
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| Relations |
| BioSample |
SAMN37798996 |
| SRA |
SRX22079181 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7838944_S4_Input_UN_FLAGx3BAP1_paired_bowtie2_res1_norm1e7.bw |
555.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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