Hiseq ten

ATAC-seq was performed with the Illumina Nextera DNA Preparation kit (FC-121-1030, Illumina)^{93 (link)}. Briefly, 50,000 live cells were freshly isolated and resuspended in lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl₂, 3 mM MgCl₂, 0.1% NP40) and pelleted. The cell pellet was resuspended in transposase reaction mix (12.5 μl 2× TD buffer, 1.25 μL transposase, 11.25 μL nuclease-free water) and incubated at 37 °C for 30 min. Fragmented DNA was purified using Zymo DNA concentrator kit (D4014, Zymo Research) and library was generated by PCR using KAPA HiFi PCR master mix (KK2602, Kapa Biosystems). An appropriate number of PCR cycles was determined by qPCR PCR cleanup of libraries was performed using AMPure beads (A63881, Beckman Coulter) at a 1.2 ratio. Libraries were then sequenced on Illumina HiSeq × Ten with paired-end reads. Reads were mapped using Bowtie2 (v2.3.5) followed by removing PCR duplicates using Picard (v2.19.0). Peaks were called using MACS2 (v2.1.2).

We performed WGS (5× coverage) using Novogene. We sequenced the genomic DNA libraries on an Illumina HiSeq × Ten and NovaSeq platform (San Diego, CA, USA) (150-bp paired-end reads). We adopted the GotCloud pipeline^{49 (link)} to detect variants from our low-pass WGS data. In brief, we subjected sequencing data to FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality control and Trimmomatic^{50 (link)} to trim and filter low-quality reads. We mapped clean data to the GRCh37 reference genome containing decoy fragments using BWA-mem. We conducted subsequent analysis by subjecting data to the GotCloud pipeline with data processing and variant detection using the default settings^{18 (link)}. We then subjected the clean genotype files to Beagle^{51 (link)} for genotyping refinement. For Chinese WGS cohort 2, we also used Thunder^{52 (link)} for genotyping refinement after Beagle processing.

Total RNA was extracted from the tissue using the TRIzol^® Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions, and genomic DNA was removed using DNase I (TaKara, Kusatsu City, Japan). Next, RNA quality was determined by a 2100 Bioanalyser (Agilent, Santa Clara, CA, USA) and quantified using an ND-2000 (NanoDrop Technologies, Wilmington, DE, USA). Only high-quality RNA (OD 260/280 = 1.8–2.0, OD 260/230 ≥ 2.0, RIN ≥ 6.5, 28 S:18 S ≥ 1.0, ≥100 ng/μL, ≥2 μg) was used to construct sequencing library. RNA-seq library was prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA, USA). After being quantified by the TBS380, a paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq×TEN (2 × 150 bp read length). The data generated by the Illumina platform were analyzed using bioinformatics analysis. All analyses were carried out on the Majorbio Cloud Platform (www.majorbio.com (accessed on 5 September 2021)) from Shanghai Majorbio Bio-pharm Technology Co., Ltd. The major software and parameters are as follows. High quality reads in each sample were aligned to a genome using Bowtie2 v.2.3.5.1 [26 (link)]. Quantify gene and isoform abundances were calculated using RSEM v.1.3.3 [27 (link)]. Then differential expression analysis was performed by SARTools v.1.9 [28 (link)].

Total RNA was extracted from each cell group using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s instructions. RNA sample quality was assessed using Nanodrop 2000 (ThermoFisher) and BioAnalyzer 2100 (Aglient Technologies). The RNA-seq libraries were sequenced using a TruSeq Stranded mRNA library preparation kit [(Illumina, San Diego, CA, United States)] and constructed as well as sequenced using HiSeq × Ten (Illumina) by BioMarker (Beijing, China). The RNA-seq reads were trimmed, filtered, and quality-controlled using FastQC (Babraham Institute) tools. Reads per kilobase per million mapped reads (RPKM) were then calculated using Hisat2 (Kim et al., 2015 (link)).