Mgiseq 2000

Cell suspensions obtained from eight fractions were used for plasmid isolation followed by PCR amplification with the primer pair 5′-GTGCTCAAGCTTTAATACGACTCACTATAGGG-3′ and 5′-GCTCTCGTCCGTCTCTTTC-3′. The PCR products were purified and checked with electrophoresis in 2% agarose gel. The obtained amplicons were used for the library preparation with the MGIEasy Universal DNA Library Prep (MGI Tech, Wuhan, China). DNA libraries were sequenced on the MGIseq-2000 (MGI Tech) utilizing the DNBSEQ-G400RS High-throughput Sequencing Set (cat. FCL PE100, MGI Tech, Wuhan, China) in the SB RAS Genomics Core Facility (ICBFM SB RAS, Novosibirsk, Russia).

Total RNAs were isolated from iPSCs treated with 5 μg/mL and 10 μg/mL ECR using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and RNA quality was assessed by 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA). The library was prepared using the MGIEasy RNA Directional Library Prep Kit and sequenced using MGISEQ-2000 (MGI Tech, Shenzhen, China) to generate 100-bp paired-end reads. Reads were trimmed using Trim Galore to remove adapter sequences and reads with low sequence quality. High-quality sequence reads were mapped to the human genome (hg38), and the expression levels of mRNAs were quantified using the DESeq2 [19 (link)]. The differences in expression levels between ECR treatment and control groups, in terms of the rate of change (log transformation) and statistical significance (false discovery rate; FDR < 0.01), were analyzed using the edgeR package [20 (link)] in R. ClueGO [21 (link)], a Cytoscape plug-in tool, was used to functionally grouped gene ontology (GO) and pathway annotation networks.

Deep WES were performed on two replicates of diluted standard samples, labeled as groups A and B. Sequencing platform was MGISEQ‐2000 (MGI Tech) with PE150 chemistry. The deduped coverage depth for all samples was above 1000×. Sequencing reads were aligned to genome (hs37d5) by Sentieon BWA.³² Somatic SNVs/INDELs were called by Sentieon TNscope and annotated by VEP (ensembl release 93). Mutations with <0.01 allele frequency (AF), >.01 PV (p‐value by Fisher's exact test of the number of reads supporting the reference and alternate alleles in the tumor and normal samples), ≤8.49 TLOD (log odds that the variant is present in the tumor sample relative to expected noise), ≤26.07 NLODF (log odds that the variant is not present in the normal sample, not a germline variant, given the AF in the tumor sample), >2.74 SOR (symmetric odds ratio to detect strand bias), ≤ −0.39 or >0 MQRankSumPS (Z‐score of Alt vs. Ref read mapping qualities per sample), or mutations in repetitive sequences were filtered out (https://support.sentieon.com/appnotes/out_fields/).

For RNA sequencing, total RNA was extracted from liver or cell samples using TRIzol reagent (T9424; Sigma-Aldrich; St. Louis, MO, USA) as indicated above. Then 200 ng of RNA input per sample and a MGIEasy RNA Library Prep Kit (1000006383; MGI Tech; Shenzhen, China) were used to construct cDNA libraries, according to the manufacturer’s instructions. Single-end libraries were sequenced using MGISEQ 2000 (MGI Tech; Shenzhen, China). For data processing, HISAT2 software (version 2.1.0)^{54 (link)} was used to map the sequences from clean reads to Ensembl mouse (mm10/GRCm38) reference genomes. SAMtools software (version 1.4)^{55 (link)} was used to sort and convert the mapped reads to the Binary Alignment Map (BAM) files. StringTie software (version 1.3.3b)^{56 (link)} was used to calculate the fragments per kilobase of exon model per million mapped fragments (FPKM) values of each gene. DESeq2 software (version 1.2.10)^{57 (link)} was used to analyze differential gene expression. Genes with a fold change greater than 1.5 and corresponding adjusted p values less than 0.05 were identified as DEGs.