title: GSM2981006: Smad23DKO_EpiLC_clone_KT15.1F6_differentiation1 (ATAC-seq); Mus musculus; ATAC-seq
library_construction_protocol: Two technical replicates of 75,000 wild type or Smad2/3 DKO single-cell suspensions of day 2 epiblast-like cells were harvested and washed with PBS (w/o MgCl2/CaCl2). Three independent differentiations per genotype were used a biological replicates. Nuclei were isolated using 50ul of cold lysis buffer (10mM Tris-HCl, pH7.4, 10mM NaCl2, 3mM MgCl2, 0.1% IGEPAL CA-630), spun down immediately at 500g for 10min, 4C, washed once with 50ul cold PBS buffer and spun down (500g for 10min, 4C). Supernatant was discarded and the pellet resuspended in transposition reaction mix (for each reaction: 2.5ul Tn5 transposase (Illumina, Cat#FC-121-1030), 25ul 2xTD buffer (Illumina, Cat#FC-121-1030), 22.5ul nuclease-free water) and incubated at 30C for 30min before purification of tagmented DNA using a Qiagen MinElute Kit (Qiagen, Cat# 28004). 100ng genomic DNA of wild type day 2 epiblast-like cells were also treated with transposition reaction mix and treated as above as a control reaction (Tn5control). ATAC-seq libraries were prepared by PCR amplification using custom Nextera index primers Ad2.1-2.13 (Supplementary Table 1 in Buenrostro et al. (2013)) with NEBNext High-Fidelity 2x PCR master mix (NEB, Cat#M0541) with 11 cycles and purified using a Qiagen MinElute Kit (Qiagen, Cat# 28004) prior to sequencing (75bp paired-end) on the Illumina HiSeq4000 platform.
attributes.tag: GEO Accession
attributes.value: GSM2981006