title: GSM3061417: revitalized MSCs ATAC-Seq rep1; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-seq was performed as previously described. 2,700-5,000 MSCs were sorted into 4°C 1× PBS and washed in cold 1× PBS, lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% Igepal CA-630) to obtain nuclei. Immediately after lysis, nuclei were spun at 500 g for 10 min using a refrigerated centrifuge. To perform Transpose reaction, immediately following the nuclei prep, the nuclei pellet was resuspended in the transposase reaction mix (2.5 μl 2× TD buffer, 0.25 μl transposase (Illumina) and 2.25 μl nuclease-free water). The transposition reaction was carried out for 30 min at 37 °C. We amplified the library fragments using 5 μl of transposed DNA, 10 μl of nuclease-free H20, 25 μl of NEBnext High-Fidelity 2× PCR master mix (New England Biolabs), 2.5 μl of 25 μM custom PCR primers 1, and 2.5 μl of 25 μM Barcode PCR primers 2 (primer sequences are previously described), using the following PCR conditions: 72°C for 5 min; 98°C for 30 s; and thermocycling at 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. We amplified the libraries for a total of 13 or 17 cycles. The appropriate number of cycles for PCR amplification was determined through qPCR as described. After the initial 5 cycles additional 8 or 12 cycles were performed depending on the yield measured by qPCR. All libraries were sequenced on the Illumina Hiseq 2500 with 100 bp paired-end reads.
attributes.tag: GEO Accession
attributes.value: GSM3061417