|Public on Jun 15, 2016
|cell line: G1E ER4
treatment: estradiol 13h
transgene: stably expressing YFP-MD
nocodazole: 1.5h release
promoter anchor: Myc promoter
raw file ids merged: 825_979_973
|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.
|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.
|After cell fixation with 1% formaldehyde for 10min and cell sorting as described above, Capture-C was performed with a double-capture procedure (Davies et al. 2016). Chromatin was digested using DpnII. We used biotin labelled DNA oligos to pull down the target restriction fragments.
Capture-C libraries were sequenced on Nextseq500 with 2x75 bp paired end sequencing.
|Illumina NextSeq 500
|Myc promoter anchor
|Library strategy: Capture-C
The raw reads were processed using published scripts (https://github.com/telenius/captureC/releases).
Custom scripts in R to normalize data by the total number of reads representing fragments ligated to the anchor region in the library.
For each time point and anchor, used custom scripts in R to combine reads representing fragments ligated to the anchor region in the library into one .bedgraph file merged across restriction digestion and/or capture replicates.
Supplementary_files_format_and_content: .bedgraph files containing coverage of reads (read counts of ligation products to given anchor region, per thousand total reads that represent ligation products to given anchor region).
|Jun 13, 2016
|Last update date
|May 15, 2019
|Pennsylvania State University
|303 Wartik Lab
|A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition [CaptureC]
|A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition