GSM9821500: scRNA-seq of primary colon tumor from patient S7; Homo sapiens; RNA-Seq
After harvested, tissues were washed in ice-cold PBS (Hyclone, SH30256.01) and dissociated using SeekMate Tissue Dissociation Reagent Kit A Pro (SeekGene, K01801301) or SeekMate Tissue Dissociation Kit C (SeekGene, K01501) from SeekGene as instructions. DNase I (Sigma, 9003-98-9) treatment was optional according to the viscosity of the homogenate. Cell count and viability was estimated using Fluorescence Cell Analyzer (Countstar® Rigel, S2) or SeekMate Tinitan Fluorescence Cell Counter (SeekGene, M002C) with AO/PI reagent after removal of erythrocytes (Solarbio, R1010) and then debris and dead cells removal was performed if needed (Miltenyi, 130-109-398/130-090-101). Finally, fresh cells were washed twice in the RPMI1640 (Gibco, 11875119) and then resuspended at 1x10^6 cells per ml in RPMI1640 and 2% FBS (Gibco, 10100147C). Single-cell RNA-Seq libraries were prepared using SeekOne® Digital Droplet Single Cell 3' library preparation kit (SeekGene, Catalog No. K00202). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then added to the sample well in SeekOne® chip S3. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip S3. After emulsion droplet generation, reverse transcription was performed at 42°C for 90 minutes and inactivated at 85°C for 5 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired, A-tailed and ligated to sequencing adaptor. Finally, the indexed PCR was performed to amplify the DNA representing 3' polyA part of expressing genes which also contained Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were cleanup with VAHTS DNA Clean Beads (Vazyme, N411-01), analyzed by Qubit (Thermo Fisher Scientific, Q33226) and Bio-Fragment Analyzer (Bioptic, Qsep400). The libraries were then sequenced on Illumina NovaSeq 6000 with PE150 read length.