GSM8647470: Cortical_neuron_ATAC_seq_22CAG_rep2_1; Homo sapiens; ATAC-seq
For Atac-seq, We used 50,000 nuclei for the transposase reaction, following the method described previously. Subsequently, samples were purified with the DNA Clean & Concentrator–5 Kit (Zymo Research), and then PCR-amplified using Nextera indexing primers (Illumina). The number of PCR cycles were optimized by qPCR to avoid over amplification of the libraries. The final libraries were purified using 1x PCRClean DX beads (Aline Biosciences). The enrichment of accessible chromatin regions in the libraries were assessed by qPCR using primers mapped to open chromatin region and gene desert regions. For ChIP-seq data 1X106 cells were incubated in lysis buffer (50 mM Tris-HCL pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% Na- deoxycholate, and 5 mM CaCl2 supplemented with protease inhibitors and 10 mM sodium butyrate for K27ac ChIP) for 20 min on ice. The chromatin was digested to 1-5 nucleosomes using Micrococcal Nuclease (NEB # M0247S), at concentration determined by titration experiments, for 10 min at 37oC. To terminate the digestion, 20 mM EDTA was added to the samples. The antibody was added and incubated overnight at 4oC. On the next day, 25 µl of protein G Dynabeads (Thermo Scientific # 10004D,) was added and the samples rotated for 2 hr at 4oC. The beads were washed 6 times: 2 washes with RIPA buffer (10 mM Tris-HCl pH8, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), 2 washes with high salt buffer (10 mM Tris-HCl pH8, 1 mM EDTA, 360 mM NaCl, 1% Triton X- 100, 0.1% Na-deoxycholate, 0.1% SDS), 2X washes with LiCl buffer (10 mM Tris-HCl pH8, 1  mM EDTA, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% Na-deoxycholate) and 1 wash with TE (10 mM Tris-HCl pH8, 1 mM EDTA). The DNA was eluted by incubation of the beads in elution buffer (10 mM Tris-HCl pH8, 5 mM EDTA, 300 mM NaCl, 0.1% SDS, 50 µg Proteinase K) for 1 hr. at 62 and purified with 1X PCRClean DX beads (Aline Biosciences). For Atac-seq, samples were purified with the DNA Clean & Concentrator–5 Kit (Zymo Research), and then PCR-amplified using Nextera indexing primers (Illumina). The number of PCR cycles were optimized by qPCR to avoid over amplification of the libraries. For ChIP-seq, The sequencing libraries were constructed using the NEBNext®Ultra™ II DNA Library Prep Kit for Illumina (NEB # E7645S), followed by analysis using Fragment Analyzer™ instrument (Advanced Analytical), and quantification by a qPCR-based method (KAPA Library Quantification Kit for Illumina Sequencing Platforms)