 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 17, 2021 |
| Title |
Input-Noc2h |
| Sample type |
SRA |
| |
|
| Source name |
G1E-ER4 cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: erythroid
time point: 2h
|
| Treatment protocol |
Cells were synchronized to pro-metaphase with 200ng/ml nocodazole for 7-8.5h. For post-mitotic enrichment, cells were released from nocodazole for 0, 60min, 120min and 240min for pro-meta, early-G1, mid-G1 and late-G1 respectively.
|
| Growth protocol |
G1E-ER4 is a subline of the GATA1-null proerythroblast cell line G1E (Weiss et al, Mol. Cell. Biol. 17, 1642–51 (1997).). Cells were cultered in IMDM medium, supplemented with 15% FBS, penicillin/streptomycin, kit ligand, monothioglycerol and Epoetin alpha at 37 degrees with 5% CO2 (as described in Weiss et al., 1997).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) was performed using anti-RNA Polymerase II antibody (Cell Signaling, 14958) as described previously 1. Briefly, following sorting, cells were re-suspended in 1ml pre-cooled Cell Lysis Buffer supplemented with protease inhibitors (PI) and PMSF for 20min on ice. Nuclei were pelleted and re-suspended in 1ml Nuclear Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS, fresh supplemented with PI and PMSF) for 10min on ice. 0.6ml IP dilution buffer (20mM Tris pH 8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS, fresh supplemented with PI and PMSF) was added followed by sonication (Epishear, Active Motif, 100% amplitude, 30s ON and 30s OFF) for 45min. Samples were pelleted at 15000rpm for 10min at 4℃ to remove cell debris. Supernatant was supplemented with 3.4ml IP dilution buffer fresh supplemented with PI and PMSF, 50g isotope-matched IgG and 50l protein A/G agarose beads (A:G = 1:1, ThermoFisher 15918014 and ThermoFisher 15920010) and rotated at 4℃ for 8h to preclear the chromatin. 200μl, chromatin was set aside as input chromatin. Precleared chromatin was then incubated with 35μl A/G agarose beads (A:G = 1:1) pre-bound with anti-RNA PolII antibody (5μg/IP) at 4℃ for overnight. Beads were washed once with IP wash buffer I (20mM Tris pH 8, 2mM EDTA, 50mMNaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20mMTris pH 8, 2mM EDTA, 500mMNaCl, 1% Triton X-100, 0.01% SDS), once with IP wash buffer II (10mMTris pH 8, 1mM EDTA, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate) and twice with TE buffer (10mM Tris pH 8, 1mM EDTA pH 8). All washing steps were performed on ice. Beads were moved to RT and eluted in 200μl fresh made Elution Buffer (100mM NaHCO3, 1%SDS). 12μl of 5M NaCl, 2μl RNaseA (10mg/ml) was added to IP and input samples and incubated at 65℃ for 2h, followed by addition of 3μl protease K (20mg/ml) and incubated at 65℃ overnight to reverse crosslinking. Finally, IP and input samples were supplemented with 10ul of 3M sodium acetate (pH 5.2), and DNA was purified with QIAquick PCR purification kit (QIAGEN 28106).
ChIP-seq libraries were constructed using the Illumina’s TruSeq ChIP sample preparation kit (Illumina, catalog no. IP-202-1012). Libraries were size selected using the SPRIselect beads (Beckman Coulter, catalog no. B23318) before PCR amplification. Libraries were then quantified through real-time PCR with the KAPA Library Quant Kit for Illumina (KAPA Biosystems catalog no. KK4835). Finally, libraries were pooled and sequenced on a Illumina NextSeq 500 platform using Illumina sequencing reagents.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Sequencing reads were mapped to mm9 using Bowtie2 (v2.2.9) with default parameters. Aligments with MAPQ score smaller than 10 were discarded. Duplicates were removed using Samtools (v0.1.19), and in addition, reads on mitochondria, contigs and blacklist regions were removed. BAM files were converted to BED files using Bedtools (v2.27.1). Peaks were called using MACS2 (v2.1.0) over corresponding input controls, with default parameters and 0.01 q-value cutoff.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files and peak files for each biologica replicate is provided
|
| |
|
| Submission date |
Mar 03, 2021 |
| Last update date |
Jun 17, 2021 |
| Contact name |
Yemin Lan |
| Organization name |
University of Pennsylvania
|
| Street address |
3400 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE168168 |
CTCF and transcription orchestrate chromatin structure re-configuration after mitosis [ChIP-seq] |
| GSE168251 |
CTCF and transcription orchestrate chromatin structure re-configuration after mitosis |
|
| Relations |
| BioSample |
SAMN18128723 |
| SRA |
SRX10229689 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
