GSM7987368: scRCAT_seq2_ES_SIRV_1; Homo sapiens; RNA-Seq
hESCs: Digest hESCs cells with Accutase at 37℃ for 15-30 min, remove the culture medium after centrifugation, resuspend the cells in PBS containing 0.04% bovine serum albumin to obtain a single-cell suspension.  Organoids: On day 45, we dissociated these organoids into single-cell suspensions by treating them with Accutase at 37°C for 30 minutes. The suspensions were then filtered through a 35-μm cell strainer, and the cells were resuspended in PBS containing 0.04% bovine serum albumin. hESCs:  The scRCAT-seq2 method encompasses the following steps: (1) Labeling the ends of individual RNA/cDNA molecules with a tag (barcode); (2) Creating a circular cDNA through end-to-end ligation; (3) Performing circular amplification to generate multiple copies of full-length cDNA, each with an identical tag; (4) Fragmenting the full-length cDNA by randomly inserting Tn5 transposes around the ligated ends; (5) Constructing libraries and conducting paired-end sequencing on the resulting fragments; (6) Integrating short reads with the same molecule tag to identify key features of full-length transcripts, such as transcription start sites (TSS), transcription end sites (TES), and specific exons (Detailed instructions can be found in the Methods section of the article). Organoids:  We used the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit from 10x Genomics, following the manufacturer's protocol to generate cDNA and scATAC library. 10 μL of cDNA is mixed with 1 μL of 10 µM read1 primer, 1 μL of 10 µM TSO-read1 primer, 15 μL of KAPA HiFi HotStart ReadyMix (2x), and the volume is supplemented with Nuclease Free Water to reach 30 μL. The reaction proceeds as follows: 98 ℃ for 3 min, 6-10 cycles of 98 ℃ for 20 s, 60 ℃ for 20 s, and 72 ℃ for 90 s, followed by 72 ℃ for 5 min. After purification using 1x AMPure XP beads, the resulting product is eluted with 10 μL Nuclease Free Water to obtain cDNA suitable for scRCAT-seq2. The TSS and TES library construction can be performed by replacing the corresponding primers in the 10x Genomics Chromium protocol.