title: GSM6437159: FL_nan_atac_rep1; Mus musculus; ATAC-seq
library_construction_protocol: Fetal livers were dissected from E13.5 mouse embryos and mechanically dispersed into single cells by gentle pipetting and straining through a 70µM filter into ice-cold PBS containing 2% FBS. Primary fetal liver cells were crosslinked in 1% formaldehyde and cell pellets were stored at -80ºC. Frozen cells were thawed and resuspended in MNase lysis buffer (0.2% NP-40, 10mM NaCl, 10mM Tris pH 8.0) and kept on ice for 15 mins. Cells were pelleted and resuspended in MNase lysis buffer with 1mM CaCl2, 40 units Micrococcal nuclease (MNase) and incubated at 37ºC for 15 minutes. The MNase reaction was stopped by adding EDTA to a final concentration of 2mM and nuclei were pelleted by centrifugation. The pellet was resuspended in 1X RIPA buffer with 1X Protease Inhibitor Cocktail and sonicated using a cup-horn sonicator (Ultrasonic VC505) at 80 Amplitude for 50 cycles with pulses of 40s on and 50s off. The lysed and fragmented chromatin was cleared by centrifugation and 5%v/v was stored at -80ºC as input. 5-10µg of antibody (depending on the titer) was added to the lysate along with a control IgG (for enrichment estimation) and antibody-lysate mixture was incubated at 4ºC for 16h (overnight). The lysate-antibody mixture was then incubated with Protein-A or Protein-G Dynabeads, depending on the antibody serotype, for 2h at 4ºC. Immunoprecipitated complexes on Dynabeads were then washed three times in 1X RIPA with protease inhibitors, and three times in wash buffer containing 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl, and protease inhibitors. This was followed by three washes in wash buffer containing 1% NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris pH 8.0, and 250 mM LiCl. Finally, beads were washed three times in 1X TE buffer containing 10mM Tris pH 8.0 and 1mM EDTA. Elution was performed by adding elution buffer containing 10mM Tris pH 8.0, 1mM EDTA, 200mM NaCl and 1% SDS and incubating at 65ºC in a Thermomixer (Eppendorf Cat# 5382000023) with gentle mixing for 20 mins. Eluate containing ChIP DNA and input chromatin was removed and incubated at 65ºC with gentle mixing overnight for reverse crosslinking. DNA was treated with 0.1µg/µl Proteinase K for 1h at 55ºC followed by purification using Phenol:chloroform extraction and ethanol precipitation. ATAC-Seq was performed exactly as in (Buenrostro et al., 2013; Buenrostro et al., 2015). ChIP-Seq libraries were made using a Neb Next DNA Ultra II DNA library preparation kit following manufacturer's instructions. Libraries were quantified using Qubit fluorometer (Invitrogen) and library quality was assessed using a high-sensitivity DNA kit on Agilent Bioanalyzer, followed by Illumina next generation sequencing with single-end 100bp reads