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| Status |
Public on Jun 15, 2016 |
| Title |
645_Async_Pol2_Rep2 |
| Sample type |
SRA |
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| Source name |
645_Async_Pol2
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| Organism |
Mus musculus |
| Characteristics |
cell line: G1E ER4
treatment: estradiol 13h
transgene: stably expressing YFP-MD
nocodazole: asynchronous
antibody: anti-Pol II N-20 (Santa Cruz, cat# sc899)
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| Treatment protocol |
Except where indicated in the text as uninduced, we induced cells to mature with 100nM estradiol to activate GATA1-ER. During estradiol induction, we simultaneously treated cells with nocodazole (200ng/ml) for 7h-13h, washed once, and replated into fresh medium lacking nocodazole for varying times (40min-360min), ensuring all samples are exposed to estradiol for the same duration of 13h. We fixed cells with 1% formaldehyde, stained with DAPI, and sorted on a BD FACSAria based on YFP-MD and DAPI signal.
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| Growth protocol |
G1E erythroblasts were previously derived through deletion of GATA1 in mouse embryonic stem cells, followed by in vitro differentiation (Weiss, Yu, and Orkin 1997). We cultured a sub-line of G1E cells, G1E-ER4, in which GATA1-ER was retrovirally transduced (referred to in main text as “G1E GATA1-ER"), as previously described (Weiss, Yu, and Orkin 1997). We retrovirally transduced G1E-ER4 cells with the YFP-MD construct (Kadauke et al. 2012) and sorted for a pool of YFP-positive cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
All samples, including input, were processed for library construction for Illumina sequencing using Illumina’s TruSeq ChIP Sample Preparation Kit (Illumina cat# IP-202- 1012). In brief, DNA fragments were repaired to generate blunt ends, purified using Agencourt AMPure XP Beads (Beckman Coulter cat# A63881), and a single “A” nucleotide was added to each end. Double-stranded Illumina adaptors were ligated to the fragments. Ligation products were purified using Agencourt AMPure XP Beads, and subject to size selection using SPRIselect Beads (Beckman Coulter cat# B23318)in which both a left side size selection was performed at 0.9x volume, and a right side size selection was performed at 0.6x volume according to manufacturer’s specifications. Library fragments were then amplified for 16 cycles of PCR and products were purified using Agencourt AMPure XP Beads. Constructed libraries were run on the Agilent Bioananlyzer 2100 (Agilent Technologies) using either the DNA 7500 kit (cat# 5067-1504) or the High Sensitivity DNA kit (cat# 5067-4626) as appropriate to determine the average size and confirm the absence of unligated adaptors. The mean library size is approximately 330 bp. The ChIP-seq libraries were quantitated by qPCR using the Kapa SYBR FAST Universal kit (Kapa Biosystems) according to the Illumina’s Sequencing Library qPCR Quantification Guide. Libraries were multiplexed and sequenced on the Illumina HiSeq 2000 using Illumina’s kits and reagents as appropriate. We sequenced 3 biological replicates for the 0min, 60min, 90min, 180min, and 360min time points; two biological replicates for 240min time point; and one replicate for 40min time point.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Rep2
async_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
522_645_775
645
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| Data processing |
Basecalls using bcl2fastq-1.8.4, and parameters --no-eamss --mismatches 1
Mapping to reference genome mm9 canon with Bowtie 1.0.0 using parameters --chunkmbs 1024 -y -n 2 --best -k 1 --maxbts 800 -l 28 -e 80 --sam-nohead --sam
Wiggle and peaks called using MACS with parameters --format BAM --gsize 1870000000 --tsize 36 --bw 120 --mfold 12 --wig --space 1
Filter blacklist regions from peaks, and convert the *peaks.xls file from MACs to broadpeak format (see UCSC Genome Browser for format specs)
R scripts for further steps that generate additional processed tabular files in SupplementalTables.zip are available at the following repository: https://chsiung@bitbucket.org/arjunrajlaboratory/hsiung_mitosisreactivation
Genome_build: mm9
Supplementary_files_format_and_content: .bigwig files containing read coverage (not normalized to total library size).
Supplementary_files_format_and_content: Pooled .bigWig files across biological replicates are linked as supplementary files on the Series record.
Supplementary_files_format_and_content: SupplementalTables.zip contains two tables that include Pol II RPKM's at the 5' 2.5kb region of active genes and intergenic enhancers, as well as results of principal components analysis and other analyses as described in the accompanying publication. Information on the columns are described in the README_SupplementalTablesDescription.xls file. R scripts that generate the two tables are available at the following repository: https://chsiung@bitbucket.org/arjunrajlaboratory/hsiung_mitosisreactivation
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| Submission date |
Jun 13, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ross Hardison |
| E-mail(s) |
rch8@psu.edu
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| Organization name |
Pennsylvania State University
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| Street address |
303 Wartik Lab
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE83263 |
A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition [Pol2ChIP] |
| GSE83293 |
A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition |
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| Relations |
| BioSample |
SAMN05233868 |
| SRA |
SRX1838940 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2197832_645.bigWig |
4.4 Gb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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