title: GSM3466716: MB_ATAC_rep2; Mus musculus; ATAC-seq
library_construction_protocol: Pooled brain regions were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation). The tissue was dissociated in the papain solution for 30 minutes at 37°C, with gentle trituration every 10 minutes using a sterile Pasteur pipette. Following dissociation, cells were passed through a 40µm cell strainer into a 50mL conical, centrifuged for 5 minutes at 300g, resuspended in albumin-inhibitor solution containing DNase, applied to a discontinuous density gradient, and centrifuged for 6 minutes at 70g. The resulting cell pellet was resuspended in HBSS with Mg2+ and Ca2+ and submitted to FACS. Aliquots of 50,000 EGFP+ cells were sorted directly into 300µL HBSS with Mg2+ and Ca2+ with 10% FBS for ATAC-seq. Aliquots containing ≥50,000 EGFP+ cells were sorted into kit-provided lysis buffer for RNA-seq. ATAC-seq library preparation generally follows the steps as set out in the original ATAC-seq paper with minor modifications. Aliquots of 50,000 EGFP+ cells were centrifuged for 5 minutes at 4°C and 500g, washed with 50µL of chilled PBS and centrifuged again for 5 minutes at 4°C and 500g. The cell pellet was resuspended in lysis buffer, as set out in the protocol, and cells were left to lyse for 5 minutes at 4°C before being centrifuged for 10 minutes at 4°C at 500g. The resulting nuclei pellet was transposed, as written, using the transposase from the Nextera DNA Library Preparation Kit. Following transposition, DNA was purified with the MinElute Reaction Clean-up Kit (Qiagen) and eluted in 10µL elution buffer. Libraries were amplified according to the original ATAC-seq protocol. The qPCR surveillance steps were modified such that the additional number of cycles of amplification were calculated as ¼ maximum intensity, so as to limit PCR duplication rates in the final libraries. Amplified libraries were purified with Ampure XP beads (Beckman Coulter) following the Nextera DNA Library Prep Protocol Guide. Libraries were quantified using the Qubit dsDNA High Sensitivity Assay (Invitrogen) in combination with the Agilent 2100 Bioanalyzer using the High Sensitivity DNA Assay (Agilent).
attributes.tag: GEO Accession
attributes.value: GSM3466716