 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 17, 2023 |
| Title |
Human6_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 |
SAMN22223924 |
| SRA |
SRX12575514 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5621777_Human_6_H3K27me3.bw |
37.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
