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Sample GSM6456406 Query DataSets for GSM6456406
Status Public on Aug 18, 2022
Title EB_CD71_INVEN_KO_ATAC_2
Sample type SRA
 
Source name E14TG2a.IV mES derived Erythroid cells
Organism Mus musculus
Characteristics strain: 129/Ola
cell type: Purified CD71+ erythroid cells derived from in vitro differentiation of INVEN KO mES cells into Embryoid Bodies (EBs) , sample 1 (INVEN KO: homozygous for the inversion of the alpha globin super-enhancer and encompassing both Nprl3 and Mpg genes, ~48kb and deleted for two CTCF sites at the Alpha Globin locus at positions (HS38 and HS39.5)), sample 2
Extracted molecule genomic DNA
Extraction protocol To produce CD71+ erythroid cells in vitro from differentiated mESCs (WT or mutant): 24h prior to differentiation, mESCs were induced by passaging into IMDM base media, trypsinised and plated in differentiation media (IMDM suplemented serum and other reagents and growth factors) in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x104 cells in 10 cm dishes or 250 cells per well of a 96-well plate for up to seven days without disruption. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. CD71-positive cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells (from disaggregated EBs) were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 ul per 10^7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 ul per 10^7 cells, following the manufacturer’s instructions) and bead-labelled cells were retained by LS columns. Cells were then processed for ATAC-Seq as in (Buenrostro et al, 2013).
Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013). 60000-80000 cells were used per biological replicate. Cells were lysed and nuclei were isolated prior to transposition with Tn5 transposase (Nextera, Illumina) for 30 minutes at 37°C. DNA was purified using a MinElute kit (Qiagen). Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013. ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). Due to the broad range of DNA fragment sizes found in these libraries, quantitation with the Qubit for DNA concentration was found to be highly variable and was omitted. The libraries were quantified using the universal library quantification kit (KAPA Biosystems). Samples were sequenced using 40 bp paired end reads (NextSeq platform).
The strategy was to survey regions of open chromatin in our cell population of interest (primary mouse derived erythroid cells or CD71+ EB-derived cells) as they mark regulatory genomic regions. The modified Tn5 can in one step identify open chromatin fragments, cut, and ligate adaptors. The fragments can then be multiplexed and sequenced
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina MiSeq
 
Description Genome-wide library of transposed elements
Data processing Trim_galore (to remove sequencing adaptors)
Alignment to the genome Bowtie 1 standard parameters with -m set to 2
Removal of PCR duplicates;
plot density of alignments in a moving 300bp window with an moving increment of 30 bp
Assembly: mm9
Supplementary files format and content: BigWig
 
Submission date Aug 15, 2022
Last update date Aug 18, 2022
Contact name Mira Tony Kassouf
E-mail(s) mira.kassouf@imm.ox.ac.uk
Organization name University of Oxford
Department Weatherall Institute of Molecular Medicine
Lab Doug Higgs
Street address John Radcliffe Hospital, Headly way
City Oxford
State/province England
ZIP/Postal code OX3 9DS
Country United Kingdom
 
Platform ID GPL16417
Series (2)
GSE211232 Multipartite super-enhancers function in an orientation-dependent manner [ATAC-Seq]
GSE211238 Multipartite super-enhancers function in an orientation-dependent manner
Relations
BioSample SAMN30312254
SRA SRX17065996

Supplementary file Size Download File type/resource
GSM6456406_EB_CD71_INVEN_KO_ATAC_2.bw 109.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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