GSM8842895: scRNA-seq of clear cell renal cell carcinoma with sarcomatoid differentiation; Homo sapiens; RNA-Seq
We used 10x Genomics Chromium Single Cell 3′ Reagents Kit v3 user guide to perform single cell suspension which were prepared before. Briefly, the single-cell samples were passed through a 40μm cells strainer and counted using a hemocytometer with trypan blue. Subsequently, the appropriate volume for each sample was calculated to recover 10,000 cells. With the 10x protocol, we added the single cells, the Gel Beads and the oils onto the 10x Genomics single-cell-A chip and run this machine. After droplet generation, samples were transferred onto a PCR tube and reverse transcription was performed using T100 Thermal Cycler (BIO-RAD). After the reverse transcription, cDNA was recovered using Recovery Agent provided by 10x genomics followed by a Silane DynaBead clean-up as outlined in the user guide. Purified cDNA was amplified for 10 cycles before being cleaned up by using SPRIselect beads. The samples were detected by Qubit2.0 Fluorometer (invitrogen) to determine cDNA concentration.Suitable nuclei concentrations were adjusted from the above experiment. As mentioned in the previous study (avoiding potential cell duplets), the number of target nucleus captured in each sample was 6,000. We adopted 10x Genomics 'Chromium Single Cell ATAC Reagent Kits User Guide' for library preparation (https://support.10xgenomics.com/single-cell-atac/library-prep/doc/technical-note-chromium-next-gem-single-cell-atac-v11-reagent-workflow-and-software-updates). Briefly, the ATAC Buffer (10x Genomics, 200122) and ATAC Enzyme (10x Genomics, 200123) were added to the 5 µL nuclei, which was incubated for 60 min at 37 °C. We prepared 'Master Mix', including 56.5 µL of Barcoding Reagent B (10x Genomics, 2000194), 1.5 µL of Reducing Agent B (10x Genomics, 2000087) and 2 µL of Barcoding Enzyme (10x Genomics, 2000125). After mixing the nuclei with the Master Mix, we added it to the Chromium Chip E (10x Genomics, 2000121). At the same time, we added Partitioning Oil (10x Genomics, 220088) and Chromium Single Cell ATAC Gel Beads (10x Genomics, 2000132) to the Chromium Chip E. The Chromium Chip E was placed into a Chromium Single Cell Controller instrument (10x Genomics) before attaching a 10x Gasket (10x Genomics, 3000072). Then, the Chromium Single Cell Controller Instrument could be run. The gel bead in emulsions (GEMs) were generated after the above steps. Subsequently, GEMs were incubated and cleaned by using Dynabeads (10x Genomics, 2000048) and SPRIselect (Beckman Coulter, B23318). scATAC-seq library construction eliminates the process of reverse transcription compared with scRNA-seq. PCR was performed after 'Sample Index PCR Mix' and Single Index Kit N Set A (10x Genomics, 1000212) were added into the samples. The details of PCR parameters could be referred to Chromium Single Cell ATAC Reagent Kits User Guide. DNA libraries were sequenced by NovaSeq 6000 (Illumina, San Diego, CA). Consistent with previous studies, we followed the 2 × 50 paired-end sequencing scheme: 50 bp read 1N, 8 bp i7 index, 16 bp i5 index and 50 bp read 2N. 10X Genomics Single Cell 3' v3 Reagent Kit and Chromium Single Cell ATAC Reagent Kits V1