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Sample GSM7904866

Query DataSets for GSM7904866

Status

Public on Apr 30, 2024

Title

ΔMBH-1_BAP1-3xT7_input

Sample type

SRA

Source name

Embryonic stem cells (ESCs)

Organism

Mus musculus

Characteristics

cell type: Embryonic stem cells (ESCs)
genotype: ASXL1/2 [Delta]MBH
treatment: Doxycycline

Treatment protocol

ESCs were infected with Doxycycline-inducible lentiviral pCW57.1 vector expressing T7-tagged BAP1. Cells were treated with 1 μg/mL Doxycycline for 1 day to induced robust BAP1-T7 expression.

Growth protocol

Mouse ES cells were maintained on 0.1% gelatin and cultured in 2i+LIF medium (47.75% Advanced DMEM/F-12, 47.75% Neurobasal, 0.5% N-2 supplement, 1% B-27 supplement minus Vitamin A, 1% GlutaMax, 1% Penicillin-Streptomycin, 1% EmbryoMax 2-mercaptoethanol supplemented with 1 µM MEK inhibitor PD0325901, 3 µM GSK3 inhibitor CHIR99021 HCl and 1,000 units/mL mouse LIF protein).

Extracted molecule

genomic DNA

Extraction protocol

Cells were cross-linked with 1% formaldehyde for 10 min and quenched by 125 mM glycine for 10 min. 30 million fixed cells were swelled in cold buffer containing 5mM PIPES (pH 8.0), 85mM KCl, 1% NP-40 and protease inhibitors for 20 min, and centrifuged at 2,000 g for 5 min at 4°C. Nuclei were resuspended with 3 mL TE buffer (pH 8.0), and subjected to sonication. Sheared chromatin was clarified by centrifugation at 4,000 g for 10 min at 4°C. The supernatant was transferred to a new tube and further supplemented with 150mM NaCl, 0.1% SDS, 1% Triton-X100 and protease inhibitors. 2% of the total material was set aside as input and 20 ng of spike-in chromatin (Active Motif, #53083) was added to each ChIP material. For each ChIP material, 2 - 8 µg of target antibodies and 1.5 μg of spike-in antibody (anti-H2Av, Active Motif, #61686) pre-bound with protein A Dynabeads (ThermoFisher) were added and incubated on a rotator at 4°C overnight. Beads were then collected on a magnetic rack and washed twice with 1 mL cold RIPA buffer, once with 1 mL cold RIPA buffer containing 500mM NaCl, twice with 1 mL cold LiCl buffer and once with 1 mL TE buffer. Finally, beads were eluted with 100 μL buffer containing 0.1M NaHCO3, 1% SDS, and 100 μg Proteinase K at 65°C overnight. Samples were then purified using QIAquick PCR purification kit (Qiagen) and eluted in 50 μL 10mM Tris-HCl.
Libraries were prepared according to NEB (Illumina kit) instructions. DNA were used to construct libraries using NEBNext® Ultra™ II DNA Library Prep kit with AMPure XP magnetic beads (Beckman Coulter).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Data processing

ChIP-seq reads were aligned to the mm10 genome assembly using bowtie2.
Peaks were called using SICERv2 with following configuration - For ChIP-Seq of histone modifications: Windows (200), Gaps (200), FDR (1e-3); for ChIP-Seq of MLL4 and T7 tag: Windows (50), Gaps (50), FDR (1e-10)
Assembly: mm10
Supplementary files format and content: wig files were generated using in-house script and the scores represent RPKM

Submission date

Nov 16, 2023

Last update date

Apr 30, 2024

Contact name

Kai Ge

E-mail(s)

kai.ge@nih.gov

Phone

301-451-1998

Organization name

NIH