title: GSM3754259: NS_ATAC_Fibroblastwchem_rep1; Mus musculus; ATAC-seq
library_construction_protocol: We prepared iCM, single-factor, and fibroblast samples for ATAC-seq as previously described (Buenrostro et al. 2013). Aliquots of 10,000–50,000 cells were spun down (310 RCF for 3 minutes) and washed with 200 μL of chilled PBS. Samples were lysed with 200 μL of chilled lysis buffer (20 mM Tris-HCl (pH 8.0), 85 mM KCl, 0.5% NP-40) and spun down at 500 RCF for 5 minutes. Nuclear pellets were transposed with 25 μL of Tagment DNA Buffer, 2.5 μL of Tagment DNA Enzyme (Nextera Sample prep Kit from Illumina, cat #FC-121-1030), and 22.5 μL of nuclease-free H2O. The samples were incubated at 37°C for 30 minutes and stored at −20°C. Transposed samples were purified using the QIAGEN MinElute Reaction Cleanup Kit (cat #28204). Libraries were amplified using 25 μL of NEBNext High Fidelity 2x PCR Master Mix, 1.25 μM Nextera custom primer, 1.25 μM Nextera custom primers with unique barcodes, and nuclease-free H2O. We amplified samples using the following PCR conditions: 72°C for 5 minutes; 98°C for 30 seconds; and cycled at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. Half of each sample was amplified for 12 cycles, MinElute purified and assessed by bioanalyzer for library quality. Samples concentration was quantified by Qbit before pooling. Samples were sequenced at 100PE on the Illumina HiSeq 2500 at either the Harvard FAS core or the UCSF CAT core.
attributes.tag: GEO Accession
attributes.value: GSM3754259