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| Status |
Public on Nov 20, 2023 |
| Title |
Large preB cell, WT, IKAROS, rep2 |
| Sample type |
SRA |
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| Source name |
mouse large pre-B cell (CD19+CD43+BP1+)
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse large pre-B cell (CD19+CD43+BP1+)
genotype: Wild-type (IKE5 fl/fl, Cre-)
chip antibody: IKAROS (Abcam, ab191394)
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| Treatment protocol |
To induce IkE5 deletion in the incucible IkE5 deletion large preB cells (IkE5 fl/fl, Rosa26-ERT2-Cre), 4-OHT (Sigma H7904, final concentration 0.2 μM ) or an equal volume of DMSO as control were added into the medium; and cells were harvested at Day 3 and D12 post induction.
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| Growth protocol |
Sorted large pre-B cells were cultured on OP9 stroma in DMEM ( D-5671; Sigma) supplemented with 10% FBS (2442; Sigma), 50 μM 2-mercaptoethanol, 100 μg/ml penicillin, 100 μg/ml streptomycin, 1× Glutamax (35050-062; Gibco), 10 mM HEPES (156-30-80; Gibco) and 1× sodium pyruvate (11360-070; Gibco) in the presence of 5ng/ml of IL-7. Stroma adherent large pre-B cells from WT or IKDN cultures were detached with 0.2% trypsin (EDTA free) for 2 minutes at room temperature. Large pre-B cells were ~ 99% of the cells in suspension as determined by flow cytometry.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed in 1% of fresh formaldehyde for 10 minutes at room temperature, quenched with glycine and washed twice with ice-cold PBS. Cells were re-suspended in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10 minutes at 4°C, pelleted, and then re-suspended in lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH8) for 5 minutes at 4°C. Cells were finally re-suspended in RIPA buffer (50mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Nadeoxycholate, 0.2% SDS, 0.5 mM PMSF, and 1x Protease inhibitor cocktail (Roche)) to a final concentration of 5x107 cells/ml. Chromatin was sonicated to an average size of 350bp with a Branson Sonifier 450 equipped with a micro probe. Chromatin was cleared by centrifugation at 20,000xg for 10 minutes, and then incubated with 5-10μg of antibodies pre-bound to Dynabeads protein G (Life technologies) and rotated overnight at 4°C. After extensive washing on beads, bound chromatin was eluted and de-crosslinked in 300μl of elution buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS, 0.3M NaCl) overnight at 65°C. Proteinases K was added to a final concentration of 200 μg/ml and incubated at 45°C for 2 hours. DNA was ethanol precipitated, re-suspended in TE and purified using the DNA Clean & Concentrator-5 kit (Zymo Research).
Purified ChIP DNA was end repaired, end adenylated, and then ligated with Illumina Truseq indexed adaptors. The ligated DNA was purified with AMPure XP beads (Beckman Coulter) and then amplified with KAPA HiFi DNA Polymerase (KAPA Biosystems) for 8 to 13 cycles. After amplification, the library DNA was size selected with AMPurex XP beads to 200-600 bp range, and the purified libraries were multiplexed for sequencing in house with an Illumina NextSeq 550 system.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
WTLpreB_IKAROS_R1_R2.pileup.bedgraph.gz
WTLpreB_IKAROS_R1_R2.pileup.bedgraph.gz.tbi
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| Data processing |
Read alignment was performed on the mm10 assembly with the STAR 5.02 genome alignment algorithm (--alignIntronMax 1 --alignEndsType EndToEnd) on the DNAnexus platform (https://www.dnanexus.com/)
Picard-tools 4.0.3 (Broad Institute) were used to remove PCR duplicates.
Peak calling was performed using MACS2 2.1.1 (Zhang et al., 2008) with input chromatin as control and with a q-value (minimum FDR) cutoff of 0.05. Model building with --mfold of 5, 50 was used for transcription factors, H3K4me3and H3K27ac. The –broad flag was used to identify enriched regions for spreading histone marks such as H3K4me1, H3K27ac, H3K9me3.
Normalized “pileup.bedgraphs” were used after indexing for browser visualization.
Bigwigs were obtained from the “pileup” files for visualization using bedGraphToBigWig v4.
Assembly: mm10
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| Submission date |
May 12, 2023 |
| Last update date |
Nov 20, 2023 |
| Contact name |
Katia Georgopoulos |
| E-mail(s) |
katia.georgopoulos@cbrc2.mgh.harvard.edu
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| Phone |
617-7264445
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| Organization name |
Harvard Medical School
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| Department |
CBRC
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| Lab |
Georgopoulos
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| Street address |
1st and 13th St
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| City |
Charlestown |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE232463 |
A multiscale 3D chromatin architecture that controls development of the humoral immune system is assembled by IKAROS [ChIP-seq LpreB cell] |
| GSE232490 |
A multiscale 3D chromatin architecture that controls development of the humoral immune system is assembled by IKAROS |
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| Relations |
| BioSample |
SAMN35066617 |
| SRA |
SRX20340936 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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