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A modified STRT-seq protocol was applied for single-cell RNA-seq. Briefly, sorted single cells in good condition were picked into lysis by mouth pipetting, and the scRNA-seq libraries were constructed based on STRT-seq with some modifications. cDNAs were synthesized using sample-specific 25-nt oligo-dT primer containing 8-nt barcode (TCAGACGTGTGCTCTTCCGATCT-XXXXXXXX-DDDDDDDD-T25, X representing sample-specific barcode while D standing for unique molecular identifiers (UMI).
After reverse transcription and second-strand cDNA synthesis, the cDNAs were amplified by 16 cycles of PCR using ISPCR primer and 3’ Anchor primer. Samples were pooled and purified using Agencourt AMPure XP beads . 4 cycles of PCR were performed to introduce index sequence and subsequently, 400 ng cDNAs were fragmented to around 300 bp by Covaris S2. After being incubated with Dynabeads MyOneTM Streptavidin C1 beads (Thermo Fisher, 65002) for 1 hour at room temperature, cDNA Libraries were generated using KAPA Hyper Prep Kit (Kapa Biosystems, kk8505). After adaptor ligation, the libraries were amplified by 8 cycles of PCR using QP2 primer and short universal primer. |
RNA-Seq |
TRANSCRIPTOMIC SINGLE CELL |
PolyA |
PAIRED
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