title: GSM3439283: ATAC_Seq_E14_5_Forelimb; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-seq was performed on dissected C57BL/6NJ E14.5 forelimb and hindlimb.  Nuclei preparation and tagmentation were performed as previously described in Buenrostro et al., 2013, doi: 10.1038/nmeth.2688., with the following modifications.  To minimize endogenous protease activity, cells were strictly limited to 5 + 5 minutes of collagenase A treatment at 37 ºC, with frequent pipetting to aid dissociation into single-cell suspensions. Following wash steps and cell lysis, 50 000 nuclei were tagmented with expressed Tn5 transposase.  Each tagmented sample was then purified by MinElute columns (Qiagen) and amplified with Q5 High-Fidelity DNA Polymerase (New England Biolabs) using a uniquely barcoded i7-index primer (N701-N7XX) and the N501 i5-index primer.  PCR thermocycler programs were 72ºC for 4 min, 98ºC for 30 s, 6 cycles of 98ºC for 10 s, 65ºC for 30 s, 72ºC for 1 min, and final extension at 72ºC for 4 min.  PCR-enriched samples were taken through a double size selection with PEG-based SPRI beads (Beckman Coulter) first with 0.5X ratio of PEG/beads to remove DNA fragments longer than 600 bp, followed by 1.8X PEG/beads ratio in order to select for Fraction A as described in Milani et al., 2016, doi: 10.1038/srep25474.
attributes.tag: GEO Accession
attributes.value: GSM3439283