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Sample GSM3711475 Query DataSets for GSM3711475
Status Public on Apr 02, 2020
Title HeLa_Mock_IP
Sample type SRA
 
Source name HeLa cells
Organism Homo sapiens
Characteristics cell line: HeLa
chip antibody: Mock IP
Growth protocol Human cell line HeLa were grown in DMEM (Invitrogen/ThermoFisher Cat #11965) supplemented with 10% FBS and 1X penicillin and streptomycin cocktail.
Extracted molecule genomic DNA
Extraction protocol Libraries were constructed with the PacBio Standard Seq (v3) protocol.
After cells were grown to ~80% confluency, they were harvested as described here (Bui et al 2012; Bui et al 2017), but with a few modifications. In short, cells were harvested, washed with PBS and PBS containing 0.1% Tween 20 (Sigma-Aldrich cat #P7949). Nuclei were released with TM2 (20 mM Tris-HCl, pH 8.0; 2 mM MgCl2; 0.5 mM PMSF) with 0.5% Nonidet P-40 (Sigma-Aldrich cat #74385). Afterwards, nuclei were washed with TM2 and dissolved in a total volume of 2 mL of 0.1 M TE (10 mM Tris-HCl, pH 8.0; 0.2 mM EDTA, 100 mM NaCl). Subsequently, chromatin was digested for 6 minutes with 0.25 U MNase (Sigma-Aldrich cat #N3755-500UN) and supplemented with 1.5 mM CaCl2. MNase reaction was quenched with 10 mM EGTA. All centrifugations were done at 1000 rpm at 4ºC. The cell or nuclei pellet was only tapped once to facilitate breaking them up. Supernatant was removed, and chromatin extracted overnight in low salt solution (0.5X PBS; 0.1 mM EGTA supplemented with a protease inhibitor cocktail (Roche cat #05056489001)
N-ChIP chromatin bound to Protein G Sepharose beads (GE Healthcare cat #17-0618-02) were washed twice with ice cold 0.5X PBS and spun down for 1 minute at 4ºC at 800 rpm. The samples were used to prepare libraries for PacBio single-molecule sequencing as described in manufacturer’s protocol (PacBio, Menlo Park, CA). Libraries were produced and loaded on ZMW chip either by diffusion or following size selection of the inserts (> 1000 bp) for all four samples.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model PacBio RS II
 
Data processing Circular Consensus Sequence (CCS) reads in fastq format were filtered to remove PacBio internal control (MG551957.1) by alignment with Bowtie v2.3.4
Filtered reads were converted to fasta, and sequence composition was examined using an in-house perl script. Alignments to alpha-satellite subunits and HORs were performed with Bowtie v2.3.4 in sensitive-local mode
Genome_build: hg19
Supplementary_files_format_and_content: TXT files include sequence composition information (mono and dinucleotide counts) for IP samples
 
Submission date Apr 04, 2019
Last update date Apr 02, 2020
Contact name David Sturgill
E-mail(s) davidsturgill@mail.nih.gov
Phone 240-760-6725
Organization name NIH
Department NCI
Lab LRBGE
Street address Building 41, Rm B622
City Bethesda
State/province MARYLAND
ZIP/Postal code 20892
Country USA
 
Platform ID GPL21311
Series (1)
GSE129351 Octameric nucleosomes are present within the inner kinetochore of human centromeres
Relations
BioSample SAMN11347520
SRA SRX5644011

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

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