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Sample GSM6685057

Query DataSets for GSM6685057

Status

Public on May 29, 2024

Title

RING1BmAID-TAF1dTAG-T7-Input-AUX-rep3

Sample type

SRA

Source name

Ring1b-mAID;Taf1-T7-dTAG

Organisms

Homo sapiens; Mus musculus

Characteristics

cell line: Ring1b-mAID;Taf1-T7-dTAG
cell type: Mouse embryonic stem cells with human T7-Scc1 HEK293T cells spike-in.
treatment: 500 µM IAA
treatment time: 4h
replicate: 3
spike-in reference: Homo sapiens
spike-in cell_line: HEK293T; Scc1-T7
antibody: none

Treatment protocol

Cell lines expressing were treated with 100 nM dTAG-13 (Tocris Bioscience) or 500 µM IAA (Sigma) for 4 hours to induce protein depletion.

Growth protocol

Mouse embryonic stem cells were grown in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with fetal bovine serum (FBS, 15% Biosera or 10% Sigma), 1x non-essential amino acids (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 1x penicillin/streptomycin (Thermo Fisher Scientific), 0.5 mM beta-mercaptoethanol (Thermo Fisher Scientific), and 10 ng/ml leukaemia inhibitory factor (produced in-house). ESCs were grown on gelatinised plates at 37⁰C and 5% CO2.

Extracted molecule

genomic DNA

Extraction protocol

5 x 10^7 ESCs were fixed with 1% formaldehyde (methanol-free, Thermo Fisher Scientific) for 10 min at 25⁰C. Fixation was quenched using glycine added to 125 mM. Cells were then pelleted at 1000xg for 5 min and washed with PBS. Cross-linked ESCs were mixed with 1 x 10^6 cross-linked HEK 293T/T7-SCC1 cells (1% formaldehyde for 10 min; a gift from Martin Houlard, Nasmyth lab). Chromatin was prepared by incubation in 1 ml FA-lysis buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1% Na-deoxycholate, 0.1% SDS, 1x PIC, 10 mM NaF) on ice for 10 min. Chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode) at 4⁰C for 23 cycles (30s on/off) to an average size of 0.5 kb. The sonicated material was pelleted at 20,000xg for 20 min, and the supernatant taken as sonicated chromatin. 300 μg chromatin was used per IP. Chromatin was diluted to 1 ml total volume per IP in FA-lysis buffer. An additional volume of diluted chromatin was taken to use as an input sample. Protein A agarose beads (Repligen) were blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA in 1x TE buffer at 4⁰C for 1 hr. Chromatin was pre-cleared with agarose beads (40 μl slurry beads per ChIP) at 4⁰C for 1 - 2 hr. The input sample was taken from the pre-cleared chromatin, and the remainder was immunoprecipitated overnight at 4⁰C with 5μl T7 (Cell Signalling, D9E1X). Antibody-bound chromatin was precipitated for 3 hr at 4⁰C using 40 μl slurry of blocked Protein A agarose beads. Washes were carried out for 5 min each at 4⁰C, using ice-cold FA-lysis buffer, FA-lysis buffer with 500 mM NaCl, 1x DOC buffer (10 mM Tris HCl, pH 8, 250 mM LiCl, 1 mM EDTA , 0.5% NP-40, 0.5% Na-deoxycholate), and 2 washes with TE buffer. DNA was eluted in elution buffer (1% SDS and 0.1 M NaHCO3). Cross-links were reversed for ChIPs and inputs at 65⁰C overnight with 200 mM NaCl and 2 μl RNase A (Sigma). Samples were then incubated with 20 μg Proteinase K for 1 hr at 45⁰C. DNA for ChIPs and inputs was purified using a ChIP DNA Clean & Concentrator kit (Zymo Research). Purified DNA was analysed using ChIP-qPCR.
cChIP-seq libraries for both ChIP and Input samples were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina, following manufacturer’s guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries were analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR quantification using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads in biological triplicate on Illumina NextSeq 500 platform.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

Input for TAF1-T7 ChIP-seq in cells treated with IAA to depleted RING1B-mAID.

Data processing

For cChIP-seq, paired-end reads were aligned to the concatenated mouse mm10 and spike-in genomes (mm10+hg19) using Bowtie2 with the ‘-no-mixed’ and ‘-no-discordant’ options. Reads that mapped more than once were discarded and PCR duplicates were removed using Sambamba.
Sequencing datasets were calibrated to the spike-in human genomes, as described previously (Fursova et al. 2019). For cChIP-seq, the number of mm10 reads were randomly downsampled to reflect the total number of hg19 reads in that sample. Furthermore, to adjust for any variation in cell mixing, each sample was adjusted using the percentage of dm6 reads relative to mm10 reads in the relevant input sample. After normalisation, read coverages for individual biological replicates were compared across regions of interest using the multiBamSummary and plotCorrelation functions from deepTools.
Assembly: mm10; hg19
Supplementary files format and content: Genome coverage tracks were generated using the pileup function from MACS2.
Supplementary files format and content: bigWig files showing the genome coverage for merged spike-in normalised biological replicates were generated using the pileup function from MACS2.

Submission date

Oct 26, 2022

Last update date

May 29, 2024

Contact name

Emilia Dimitrova

E-mail(s)

[email protected]

Organization name

University of Oxford

Department

Biochemistry