GSM9035304: ProtocolD+C,S4D4,batch2; Homo sapiens; RNA-Seq
SC-derived cells or islets were dissociated into single cells with 0.25% trypsin at 37 °C for 3-5 min, and the digestion was halted by adding 0.4 volumes of FBS. Partially dissociated SC-islets underwent a secondary trypsinization at 37°C for 3 minutes to ensure complete dissociation. Cells from both dissociation steps were pooled for further analysis. Retrieved grafts from the renal capsule were finely minced and enzymatically dissociated using 0.25% trypsin at 37°C for 5 minutes. The enzymatic reaction was halted by adding 0.4 volumes of FBS. ScRNA-seq via the Smart-seq3 method was carried out in accordance with established procedures. Briefly, individual cells were sorted into 384-well plates containing lysis buffer (0.3 μL per well). The reverse transcription of mRNA was facilitated using a 0.1 μL aliquot of reverse transcription mix, followed by pre-amplification employing a 0.6 μL aliquot of PCR mix. A diluted sample of the resulting cDNA from each cell was analyzed using quantitative PCR (qPCR) employing ChamQ™ SYBR® qPCR Master Mix (Vazyme, Q321-02) and primers targeting ACTB: forward 5'-ACAGAGCCTCGCCTTTGCC-3' and reverse 5'-GATATCATCATCCATGGTGAGCTGG-3'. Cells exhibiting cycle threshold (Ct) values under 21 for ACTB were selected for library preparation. To differentiate human cells within grafts, GAPDH expression was quantified using specific primers: forward 5'-CTCTGCTCCTCCTGTTCGAC-3' and reverse 5'-GCGCCCAATACGACCAAATC-3', considering cells as human if Ct values were below 25 and melting temperatures surpassed 85°C. Library construction was performed utilizing the TruePrep DNA Library Prep Kit (Vazyme, Cat#TD501), and sequencing was conducted on the MGIseq-2000 platform, generating 100 bp paired-end reads.     For 10x Genomics scRNA-seq, cells purified through FACS were processed on a Chromium controller. This step was followed by cDNA synthesis and library preparation in line with the protocols provided with the Single Cell 3′ Reagent Kit v3.1. The prepared libraries were then sequenced using the Illumina HiSeq 4000 system, yielding 150 bp paired-end reads.Cell suspensions were initially stained with DAPI to facilitate the sorting of live single cells utilizing the FACSAria™ Fusion flow cytometer. Subsequent to sorting, nuclei were isolated in accordance with the protocols detailed in the Chromium NEXT GEM Single Cell ATAC Reagent Kits v2 (10x Genomics). The prepared libraries were then subjected to sequencing on the NovaSeq 6000 platform, achieving 50 bp paired-end reads.     In the context of protocol A, one set of samples consisted of a composite of cells from stages S2D2, S4D3, S5D3, and S6D3, combined in a ratio of 1:2:2:3. An alternative set comprised cells from stages S3D2, S4D3, and S5D3, merged in a ratio of 1:2:3. Within the framework of protocol C, one sample amalgamated cells from stages S2D2, S3D3, S4D3, and S5D3, in a ratio of 1:2:2:3, whereas another ensemble included cells from stages S2D4, S3D3, and S4D3, in a ratio of 1:2:3.