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Sample GSM8248105 Query DataSets for GSM8248105
Status Public on May 07, 2024
Title antiSP1-dTAG-rep4
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
Data processing Adapter trimming: cutadapt
Alignment: bowtie2
File manipulation: samtools v1.16.1
File manipulation: 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
Organization name University of Connecticut
Department Center for Cell Analysis and Modeling
Street address 400 Farmington Ave
City Farmington
State/province CT
ZIP/Postal code 06030
Country USA
Platform ID GPL21697
Series (1)
GSE266489 ZNF143 binds DNA and stimulates transcription initiation to activate and repress direct target genes (ChIP-seq)
BioSample SAMN41178840
SRA SRX24438397

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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