GSM6008393: s163; Homo sapiens; RNA-Seq
For graft extraction, mice were euthanized, and the transplanted kidney was removed. SC-islets graft was peeled off the kidney, was sliced into small pieces and digested in 2mg/mL collagenase D (Sigma, 11088858001) in RPMI (GIBCO, 11875-085) for 45 min in 37 °C. Additional breaking was used by pipetting and by filtering through a 40μm cell strainer. Cells were then centrifuged and resuspended in 0.5% BSA in PBS and a magnetic mouse depletion kit (Miltenyi, 130-104-694) was used to remove residual mouse cells. All samples were loaded into Chip G per the user guide from 10x Genomics, no alterations were made at any step of the protocol (Part No. CG000315). GEMs were formed targeting 10,000 cells and reverse transcription completed immediately after. The cDNA was cleaned from the GEM reagents, amplified for a total of 11 cycles and verified via TapeStation (Agilent Technologies). Amplified cDNA was diluted and ran on the 4200 TapeStation instrument using High Sensitivity D5000 tape and reagents (Part No. 5067-5592 & 5067-5593). The amplified cDNA was fragmented, end repaired, and A-tailed followed by adaptor ligation, and PCR amplification for a total of 12 cycles with each sample receiving a unique set of dual indices (Part No. 1000215). Final libraries were diluted and ran using the High Sensitivity D5000 tape and reagents (Part No. 5067-5592 & 5067-5593) on the 4200 TapeStation (Agilent Technologies). Libraries were quantified via Kapa qPCR using the Complete Universal Kit (Part No. 07960140001, Roche Sequencing Solutions) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). Libraries were sequenced on an Illumina NovaSeq instrument using the parameters outlined in the user guide (Read1: 28 bp, i7 index: 10 bp, i5 index: 10 bp, Read2: 90 bp). 10X Chromium Single Cell 3' Library preparation and sequencing