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Sample GSM8248103

Query DataSets for GSM8248103

Status

Public on May 07, 2024

Title

antiSP1-dTAG-rep2

Sample type

SRA

Source name

HEK_CloneZD29

Organism

Homo sapiens

Characteristics

cell line: HEK_CloneZD29
genotype: ZNF143 tagged with an inducible degron tag
treatment: 50nm dTAGV-1 treatment

Treatment protocol

HEK-293T cells were harvested after either no treatment or after 30 minutes of 50nm dTAGV-1 treatment

Growth protocol

HEK293T-ZNF143-dTAG cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (R&D Systems), 1% L-glutamine (Gibco), 1% sodium pyruvate (Gibco), and 1% penicillin-streptomycin (Gibco).

Extracted molecule

genomic DNA

Extraction protocol

After treatment, cells were fixed with 1% formaldehyde for 10 minutes at 37°C and quenched with 125mM Glycine (Fisher) for 10 minutes at 37°C. Plates were moved to ice, and cells were washed and scraped into ice cold PBS containing Complete EDTA-free Protease Inhibitor Cocktail (Roche). Cells were pelleted in aliquots of 2*10^7 cells, snap frozen in liquid nitrogen, and stored at -80°C. Pellets were thawed, and cells were lysed in 1mL ChIP Lysis Buffer (0.5% SDS, 10mM EDTA, 50mM Tris-HCL pH 8.0), with protease inhibitor cocktail added fresh, for 10 minutes with rotation at 4°C. Lysates were sonicated at 70% amplitude for 15 seconds on and 45 seconds off for 4 sets of 20-minute cycles. Sonicated lysates were moved to 1.5ml tubes and clarified by centrifugation at 14,000rpm for 10 min in 4°C. 50μL of the supernatant was diluted into 760μL ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 167mM NaCl, 16.6mM Tris-HCl pH 8.0), with protease inhibitor cocktail added fresh (1*10^6 cells in 200μL). 1ml (4*106 cells) was aliquoted into each of 3 tubes with antibody (2μg anti-HA Invitrogen REF 26183, 8μg anti-SP1 Santa Cruz Biotechnology sc-17824 X, or mock IP), and incubated with end-over-end rotation at 4°C overnight. 80μL Protein A/G Magnetic Beads (New England Biolabs) per sample were washed with bead washing buffer (PBS with 0.1% BSA and 2mM EDTA) and then incubated with samples for 90 minutes with rotation at 4°C. The samples were washed once each with low salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2mM EDTA, 150mM NaCl, 20mM Tris HCl pH8.0), high salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2mM EDTA, 500mM NaCl, 20mM Tris Hcl pH 8.0), LiCl immune complex buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH8.0), and 1xTE (10mM Tris-HCl, 1mM EDTA pH8.0). Immune complexes were eluted in elution solution, (1% SDS, 0.1M sodium bicarbonate). 1μl RNase A was added to each sample for 10 min at 37°C. Proteins were digested with the addition of 5μl Proteinase K and incubated in a 65°C water bath overnight.
DNA was purified with a MinElute PCR purification kit (Qiagen), and libraries were prepared with a NEBNext Ultra II Library Prep Kit (New England Biolabs).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

NextSeq 550

Description

antiSP1-dTAG_merged_normalized.bigWig
antiSP1_ChIP_summits_final.bed

Data processing

Adapter trimming: cutadapt
Alignment: bowtie2
File manipulation: samtools v1.16.1
File manipulation: https://github.com/guertinlab/seqOutBias v1.4.0
Peak calling: macs3
File manipulation: bedtools
Assembly: hg38
Supplementary files format and content: bigWig counts files
Supplementary files format and content: peak summit counts files

Submission date

May 02, 2024

Last update date

May 07, 2024

Contact name

Jinhong Dong

E-mail(s)

jdong@uchc.edu

Organization name

University of Connecticut

Department

Center for Cell Analysis and Modeling