GSM9702308: scRNA_EHDPP; Homo sapiens; RNA-Seq
For RNA-seq, organoids exposed to EHDPP and negative control (DMSO) were collected by 500 μL TransZol Up (ET111-01, Transgen). After that, 100 μL of chloroform (M1024442500, Merck) was used before vigorous shaking before an 12,000-rpm centrifugation at 20 °C for 10 min. Top layer was aspirated and 300 μL of isopropyl alcohol (1.01040.4008, Merck) was added. And then the samples were gently shaken before an 12,000-rpm centrifugation at 20°C for 10 min. The supernatants were removed, and then 700 μL of 75% ethanol (E801077, Macklin) was used per sample before gentle shaking and centrifugation at same rpm at 20°C for 5 min. After removing the supernatants, each sample was dissolved in 30 μL of DEPC H2O (W274329, Aladdin). For scRNA-seq,For sample preparation, organoids were digested with Accumax (STEMCELL, 07921) for 10 min. Then, cells underwent washing and were subsequently resuspended in calcium- and magnesium-free 1X phosphate-buffered saline supplemented with 0.04% bovine serum albumin. For RNA-seq, RNA integrity number (RIN) was detected by Agilent 2100, and the RIN of > 7 qualified were used on RNA-seq. RNA-seq was achieved by the Beijing Genomics Institute. Poly A species were used to enrich RNA samples, and the libraries were constructed on the BGISEQ-500 platform (BGI). Sequencing depth of each library was approximate 40 million reads. For scRNA-seq, a total of 15,000 viable cells were then used for scRNA-seq library preparation via the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, Catalog No. PN-1000268) and the Chromium Next GEM Chip G Single Cell Kit (10x Genomics, Catalog No. PN-1000127) according to the manufacturer's protocol. All the libraries were pooled and sequenced via the 150 bp paired-end Illumina NovaSeq 6000 platform. Alignment and transcript quantification of the scRNA sequencing data were executed using the CellRanger count pipeline (version 8.0.1) with the mm39 reference assembly.