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Sample GSM5621773 Query DataSets for GSM5621773
Status Public on Mar 17, 2023
Title Human2_H3K27me3
Sample type SRA
 
Source name human liver
Organism Homo sapiens
Characteristics tissue: liver
chip antibody: H3K27me3 (Cell signaling technology, 9733S,Lot 16
group: old
Treatment protocol Old as treated group
Extracted molecule genomic DNA
Extraction protocol Cut&Tag was performed following guidelines in the CUT&Tag@home v.1 protocol by Henikoff et al (Henikoff et al., 2020). Briefly, nuclei were isolated from ~20mg frozen human liver tissue using Minute Detergent-free Nuclei Isolation Kit (Invent Biotechnologies). The nuclei were permeabilized with 0.1% Triton X-100 and their integrity and number was checked under a microscope using a hemocytometer. ~10,000 nuclei per sample were bound to Concanavalin A beads (Bangs Laboratories). The bead-bound nuclei were incubated with primary antibody (1 h at room temperature), secondary antibody (0.5 h at room temperature) and home-made pA-Tn5 transposome (1 h at room temperature). Targeted tagmentation was then performed by addition of buffer containing MgCl2 for 1 h at 37°C. The tagmented DNA was washed with a TAPS-EDTA wash buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA) to remove excess salt and Mg2+ ions followed by targeted release with 0.1% SDS and neutralization of SDS by Triton X-100.
The released DNA was amplified using dual-indexing primers following a PCR protocol that biases amplification of short DNA fragments. The excess primers were removed by a 1.3X SPRI bead purification (Beckman Coulter). Library quality and quantity was confirmed on a BioAnalyzer (Agilent) DNA HS chip. Equimolar amounts of each library were combined, and the pooled library was further quantified using a NEBNext Library Quant Kit (New England Biolabs).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model NextSeq 2000
 
Data processing Library strategy: CUT&Tag
Briefly, Illumina sequencing reads (~6 million paired end reads per sample) were de-multiplexed generating compressed FASTQ files by the on-board DRAGEN informatics pipeline (Illumina DRAGEN FASTQ Generation – 3.7.4) on the NextSeq 2000.
CUT&Tag analysis was performed as outlined in Zheng et al, following the protocols.io tutorial (https://dx.doi.org/10.17504/protocols.io.bjk2kkye).The qualities of the FASTQs were checked by running FASTQC/0.11.9.The reads were aligned with bowtie/2-2.4.2 using the end-to-end option.
Sam output files were then filtered to retain alignments with a minimum mapping quality of 2 using samtools/1.9. Reads mapping to Encyclopedia of DNA Elements (ENCODE) blacklist regions were removed from the analysis.
The bamCoverage option in deepTools/3.5.0 was used to generate RPKM (reads per kilobase per million mapped reads) normalized bigWig files. H3 was subtracted from H3K27me3 with bigwigCompare function of deepTools/3.5.0.
Genome_build: hg38, K-12
Supplementary_files_format_and_content: bigwig files were generated using bamCoverage in deepTools/3.5/0; Scores represent the coverage on the genome location
 
Submission date Oct 12, 2021
Last update date Jun 28, 2023
Contact name Supriyo De
Organization name NIA-IRP, NIH
Department Laboratory of Genetics and Genomics
Lab Computational Biology & Genomics Core
Street address 251 Bayview Blvd
City Baltimore
State/province Maryland
ZIP/Postal code 21224
Country USA
 
Platform ID GPL30173
Series (2)
GSE185703 A hyper-quiescent chromatin state is a barrier to productive regeneration during aging. [CUT&Tag ]
GSE185708 A hyper-quiescent chromatin state is a barrier to productive regeneration during aging.
Relations
BioSample SAMN22223920
SRA SRX12575510

Supplementary file Size Download File type/resource
GSM5621773_Human_2_H3K27me3.bw 36.7 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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