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Sample GSM4797764

Query DataSets for GSM4797764

Status

Public on Dec 31, 2022

Title

Control_H3K27me3_2

Sample type

SRA

Source name

Epidermal progenitor cells

Organism

Mus musculus

Characteristics

cell type: Epidermal progenitor cells
tissue: Epidermis
age: Postnatal day 0
genotype: Control
chip antibody: H3K27me3

Extracted molecule

genomic DNA

Extraction protocol

Basal layer epidermal progenitors were isolated from newborn mice. Briefly, P0 back skins were collected and incubated for 4-6 hours in 1.26U/mL dispase (Invitrogen) at 4ºC. The epidermis was gently peeled from the underlying dermis, dissociated by 0.25% Trypsin with 2.21mM EDTA (Corning Cellgro; Manassas, VA, USA) and washed twice with 1x PBS. Cells were stained with 1:200 Sca1-PerCP-Cy5.5 (Biolegend), 1:100 α6-integrin-FITC (eBiosciences), and 1:200 EpCAM-APC (Biolegend) for 30 minutes on ice, and washed twice with 1x HBSS prior to cell sorting. Interfollicular epidermis, enriched for epidermal progenitors, was sorted as EpCAM(+), Sca1(+), and α6-integrin(high). A total of 0.4 x 10^6 cells was used per ChIP. Prior to cell sorting, cells were stained for viability using Zombie violet (Biolegend; San Diego, CA), then cross-linked using fresh solution with a final concentration of 1% formaldehyde (Thermo Fisher Scientific; Rockford, IL) for 10 minutes at room temperature. Crosslinking was stopped by the addition of Glycine (final concentration 125mM) for 5 minutes of incubation at room temperature, followed by two washes with 1x PBS. Cells were incubated in lysis buffer 1 (50mM HEPES pH=7.5, 140mM Nacl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitor cocktail (Roche)) for 10 min on ice, then incubated for 10 min with lysis buffer 2 (10mM Tris-HCl pH=7.5, 200mM NaCl, 1mM EDTA, 0.5mM EGTA). Before ChIP, cells were resuspended in lysis buffer 3 (10mM Tris-HCl pH=8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% N-laurylsarcosine, 1% Triton X-100) and sonicated using a Bioruptor Sonicator (Diagenode, UCD-200) according to a 25x regimen of 30 seconds of sonication followed by 90 seconds of rest at 2.7ºC. Chromatin was incubated overnight at 4ºC with anti-H3K27me3 antibody as indicated in Table S7. Dynal protein G magnetic beads (Invitrogen) were added the next day and incubated for 4 hours. The beads were sequentially washed with low salt, high salt, LiCl, and Tris-EDTA buffers for 10 minutes each at 4ºC. Bound chromatin was eluted and crosslinking was reversed by overnight incubation at 65ºC, followed by RNase A (Sigma-Aldrich) and proteinase K (Roche Diagnostics) treatments. Samples were purified using ChIP DNA Clean and Concentrator kit (Zymo Research; Irvine, CA).
For high-throughput ChIP sequencing, libraries were constructed from 3ng of Purified DNA using the DNA SMART ChIP-Seq Kit (Clontech; Palo Alto, CA, USA) according to the manufacturer’s instructions. Constructed ChIP-seq libraries were sequenced on the Illumina HiSeq platform

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Data processing

The ChIP-seq reads were aligned to the mouse reference genome (mm10) and the fly genome (Dm6) using the bowtie2 software (v2.3.3.1). Uniquely aligned reads to the mouse genome were kept for downstream analysis, after duplicate reads removed by the software samtools (v1.9). Genomic regions broadly enriched with ChIP-seq singling were determined using a sliding window approach with the input as controls. Peaks consistently called in all replicates were used as the final peaks. Data from knockout samples without expected enriched signals were not used for peak calling, as they were experimental controls.
Genome_build: mm10
Supplementary_files_format_and_content: bed

Submission date

Sep 21, 2020

Last update date

Dec 31, 2022

Contact name

Deyou Zheng

E-mail(s)

deyou.zheng@einsteinmed.edu

Organization name

Albert Einstein College of Medicine

Street address

1300 Morris Park Ave