GSM7882498: placenta villi, late pregnancy snATAC, donor2 [term-atac-2]; Homo sapiens; ATAC-seq
The villi were separated from the early or term placenta and cut up after removing the blood with phosphate buffer solution (PBS). Ice cold Nuclei EZ lysis buffer (Sigma-Aldrich, NUC-101) containing RNAase inhibitor (TaKaRa, 2313B) and protease inhibitor (Sigma-Aldrich, P8340) were added to resuspend the tissue and grinded with a dounce tissue grinder (Sigma-Aldrich, D8938). The lysates were bathed in ice and lysed twice. After washing with PBS, the nuclei were splited in half and filtered through a 40 μm sieve to proceed ATAC-seq and RNA-seq, respectively. Single-nucleus RNA Sequencing. Single Cell B Chip Kit (10x Genomics, 1000074) and the nucleus suspension (600 nuclei per microliter determined by CountStar) were loaded onto the Chromium single cell controller (10x Genomics) to generate single-nucleus gel beads in the emulsion (GEMs) according to the manufacturer's protocol. In brief, single nuclei were suspended in PBS containing 0.04% Bovine serum albumin (BSA). About 16,000 nuclei were added to each channel, with 8,000 target nuclei estimated to be recovered. Captured nuclei were lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on an S1000TM Touch Thermal Cycler (Bio-Rad) at 53°C for 45 min, followed by 85°C for 5 min and held at 4°C. The cDNA was generated and then amplified, and quality was assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing). Next, single-nucleus RNA-Seq library was constructed using Single Cell 3' Library and Gel Bead Kit V3.1 according to the manufacturer's introduction. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per nuclei with a pair-end 150bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).   Single-nucleus ATAC Sequencing. Following the 10x Genomics single cell ATAC solution, by using Chromium Chip E Single Cell Kit (Product Code 1000156) and Chromium Single Cell ATAC Library & Gel Bead Kit (Product Code 1000110), the nuclei in a bulk sample were partitioned into nanoliter-scale Gel Beads-in-emulsion (GEMs), a pool of ~750,000 10x Barcodes was sampled to separately and uniquely index the transposed DNA of each nucleus. Libraries were then generated (performed by CapitalBio Technology, Beijing). The libraries were sequenced using an Illumina Novaseq sequencer with a sequencing depth of at least 25k read pairs per nuclei with a pair-end 50bp (PE50) reading strategy.