title: GSM6456404: EB_CD71_INVEN_ATAC_3; Mus musculus; ATAC-seq
library_name: GSM6456404
library_construction_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