NSG_Tu_1 (CRX1285295; CRA020963)
Single-cell RNA-Seq libraries were prepared using SeekOne\u00ae Digital Droplet Single Cell 3\u2019 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\u00ae 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\u2103for 90 minutes and inactivated at 85\u2103 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\u2019 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.