GSM9047881: patient-3; Homo sapiens; RNA-Seq
After harvested, tissues were washed in ice-cold PBS (Hyclone SH30256.01) and digested using Trypsin (Hyclone SH30042.01) 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 SeekMate Tinitan Fluorescence Cell Counter (SeekGene M002C) with AO/PI reagent after removal erythrocytes (Solarbio R1010) and then debris and dead cells removal was decided to be performed or not (Miltenyi 130-109-398/130-090-101). Finally fresh cells were washed twice in the RPMI-1640 (Gibco 11875119) and then resuspended at 1×106 cells per ml in RPMI-1640 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 were performed at 42℃ for 90 minutes and inactivated at 85℃ 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 were performed to amplified 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 X Plus with PE150 read length.