title: GSM5324899: GABA neurons ATAC-seq rep1; Homo sapiens; ATAC-seq
library_construction_protocol: ATAC-seq: Adherent cells were dissociated with TrypLE Select Enzyme and centrifuged at 500 g for 5 min at 4°C. The cell pellet was resuspended in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and centrifuged for 20 min at 500 g at 4°C. 4C-seq: Cells were dissociated to single cells with TrypLE Express and fixed in 2% formaldehyde in DPBS (Thermo Fisher) for 10 min. Glycine was added to a final concentration of 222 mM to quench the cross-linking reaction. Cells were centrifuged for 10 minutes at 400g at 4°C, the supernatant discarded, and the cell pellet resuspended in cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, cOmplete protease inhibitor cocktail, Roche) and incubated on ice for 30 minutes. To ensure complete lysis, cells were stained with methyl green pyronin (Dianova). In the case of efficient lysis, nuclei are stained blue and cytoplasm pink. Next, lysed cells were centrifuged and the pellet was washed with DPBS and resuspended in water. NEBuffer and SDS (0.3% final concentration) were added and the samples were incubated at 37°C for 1 hr while shaking at 900 rpm with occasional pipetting to break cell aggregates. Triton X-100 (Biomol) was added to a concentration of 2.5% and samples were again incubated at 37°C for 1 hr while shaking. The first round of digestion was done with DpnII (New England Biolabs) at 37°C overnight. Digest efficiency was checked on an agarose gel. DpnII was inactivated by incubating for 20 min at 65°C. Ligation was performed with T4 ligase (Thermo Fisher) overnight at 16°C and checked on an agarose gel. Samples were then decrosslinked with Proteinase K at 65°C overnight. After DNA purification with phenol-chloroform, the second round of digestion was performed with Csp6I (Thermo Fisher). Digestion was followed by overnight ligation with T4 ligase at 16°C. Samples were purified using Amicon Ultra-15 columns (Millipore) and DNA quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher). ATAC-seq: Purified samples were then amplified by PCR (maximum 12 cycles) using barcoded primers from the Nextera Index Kit (Illumina). To determine the optimal number of cycles for each sample, samples were first amplified for 5 cycles (5 min at 72°C, 30 sec at 98°C, and 5 cycles of 10 sec at 98°C, 30 sec at 63°C, and 1 min at 72°C). Samples amplified for 5 cycles were then subjected to qPCR (SYBR Green, Thermo Fisher). The additional number of cycles required was the number of cycles required to reach one quarter of the maximal fluorescence intensity in the first 5 cycles. The remainder of each sample was subsequently amplified for an additional 5-7 cycles. Amplified samples were purified with SPRI beads (Beckman Coulter) at a ratio 1:1.8 sample:beads, and fragment size was checked using the High Sensitivity DNA kit (Agilent). Libraries were sequenced on a HiSeq 4000 (Illumina), two samples per lane, 100 bp paired end reads. 4C-seq: To generate 4C libraries, we used 3.2 µg of DNA for PCR amplification divided into 16 PCR reactions, using Expand Long Template PCR System (Sigma Aldrich). PCR primers were designed using the MEIS1 promoter as a viewpoint and included TruSeq adapters. Primer genomic targets are shown in Fig. S3 and sequences in Table S1. PCR conditions were 2 min at 94°C, 30 cycles of 15 sec at 94°C, 1 min at 55°C, and 3 min at 68°C followed by 5 min at 68°C. Pooled libraries were purified using SPRI beads (Beckman Coulter) at a ratio of 1:1.8 sample:beads, and fragment size was checked using the High Sensitivity DNA kit (Agilent). The libraries were sequenced on an Illumina MiSeq.