GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2610626

Query DataSets for GSM2610626

Status

Public on Sep 15, 2017

Title

RNA_shCbx6_R2

Sample type

SRA

Source name

mouse ESCs

Organism

Mus musculus

Characteristics

cell line: E14Tg2A

Treatment protocol

Two 15-cm plates for each cell line to be tested were prepared at 70–80% confluency. Cells were trypsinized and crosslinked in 1% formaldehyde for 10 min at room temperature in a shaker. To stop the fixation reaction, 0.125 M glycine was added to the existing culture media and incubated for 5 min. Sample pellets were then washed twice with PBS 1× at room temperature. After aspirating PBS completely, crosslinked pellets were resuspended in 1.3 ml ice-cold IP buffer (1× volume SDS buffer [100 mM NaCl, 50 mM Tris-HCl, pH 8., 5 mM EDTA, pH 8, and 2% SDS] and 0.5 volume Triton dilution buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.6, 5 mM EDTA, pH 8 and 5% Triton X-100]) with proteinase inhibitors. Samples were sonicated for 12 min (30 sec ON/30 sec OFF) in a Bioruptor (Diagenode) at maximum output. After sonication, samples were centrifuged at 4°C at maximum speed for 20 min. To check chromatin size, 20 µl of the supernatant was mixed with 80 µl of 1× PBS and de-crosslinked for 3 h at 65°C in a shaker (1000 rpm), followed by PCR purification kit (Qiagen). DNA was eluted in 30 µl of water and quantified by Nanodrop. Around 800 ng were loaded in a 1% agarose gel. If chromatin was between 200–500 bp, 40 µg of chromatin was used to ChIP proteins.

Growth protocol

Wild-type (E14Tg2A) ESCs were cultured feeder-free in plates coated with 0.1% gelatin. Coating was achieved by covering the plates with gelatin 15 min at 37°C. After removing any remaining gelatin, ESCs were cultured with Glasgow minimum essential medium (Sigma) supplemented with β-mercaptoethanol, sodium pyruvate, penicillin-streptomycin, non-essential amino acids, GlutaMAX, 20% fetal bovine serum (Hyclone) and leukemia inhibitory factor (LIF).

Extracted molecule

total RNA

Extraction protocol

RNA was extracted with the RNeasy mini kit (Qiagen) following manufacturer's instruction. Genomic DNA for the HA and Cbx7 ChIPs was extracted using the ChIP-IT High sensitivity kit, from Active Motif. Genomic DNA for the Ring1B ChIP was extracted according to the protocol in the Materials and Methods. IP buffer + sonication. IP buffer = (1 volume SDS buffer [100 mM NaCl, 50 mM Tris-HCl, pH 8., 5 mM EDTA, pH 8, and 2% SDS] and 0.5 volume Triton dilution buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.6, 5 mM EDTA, pH 8 and 5% Triton X-100]) with proteinase inhibitors
Libraries were prepared according to Illumina instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

FPKM_shCTL-shCbx6_R2.txt

Data processing

ChIPseq analysis: Alignment: Sequence reads were mapped to the Mouse genome (mm9, July 2007) using BOWTIE (PubMed ID 19261174) with the option -m 1. RNAseq analysis: Alignment: Sequence reads were mapped against the mm9 mouse genome assembly using TopHat (PubMed ID 22383036) with the option ‐g 1. Cufflinks (PubMed ID 22383036) was run to quantify the expression in FPKMs of each annotated transcript in RefSeq.
Peak-calling: Peak detection was performed with MACS (PubMed ID: 18798982). ChIPseq profiles were produced by MACS in BedGraph format. Peaks were reported in BED format.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP_HA-Cbx6_R1.bdg.gz (BedGraph, genome-wide ChIP profile)
Supplementary_files_format_and_content: ChIP_HA-Cbx6_R2.bdg.gz (BedGraph, genome-wide ChIP profile)
Supplementary_files_format_and_content: ChIP_HA-Cbx6_Control.bdg.gz (BedGraph, genome-wide ChIP profile)
Supplementary_files_format_and_content: ChIP_Cbx7.bdg.gz (BedGraph, genome-wide ChIP profile)
Supplementary_files_format_and_content: ChIP_Cbx7_IgG.bdg.gz (BedGraph, genome-wide ChIP profile)
Supplementary_files_format_and_content: ChIP_Ring1b.bdg.gz (BedGraph, genome-wide ChIP profile)
Supplementary_files_format_and_content: ChIP_Ring1b_IgG.bdg.gz (BedGraph, genome-wide ChIP profile)
Supplementary_files_format_and_content: ChIP_HA-Cbx6_R1_peaks.bed (BED, signal enriched regions)
Supplementary_files_format_and_content: ChIP_HA-Cbx6_R2_peaks.bed (BED, signal enriched regions)
Supplementary_files_format_and_content: ChIP_Cbx7_peaks.bed (BED, signal enriched regions)
Supplementary_files_format_and_content: ChIP_Ring1b_peaks.bed (BED, signal enriched regions)
Supplementary_files_format_and_content: RPKMs_shCtlR1R2-shCbx6R1R2.txt (RPKM values control r1, control r2, shCbx6 r1, shCbx6 r2.)

Submission date

May 09, 2017

Last update date

May 15, 2019

Contact name

Enrique Blanco

E-mail(s)

enrique.blanco@crg.eu

Phone

+34 93 316 01 00

Organization name

Center for Genomic Regulation (CRG)