OEX011324
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Transcriptomic single cell |
Transcriptomic single cell |
Single-cell RNA-seq library construction was performed according to the instructions from the modified Smart-seq2 protocol (Picelli et al., 2014). In brief,
megakaryocytes were sorted by FACS according to CD41a+CD42b+ phenotype and transferred into lysis buffer by manual picking.
Cells were vortexed and incubated at 72C for 3 minutes to ensure complete lysis. Reverse transcription PCR with template switch
oligo (TSO) primer, and the cDNAs were amplified by 14 to 16 PCR cycles with 30 P2 primer and IS primer. Agencourt AMPure XP
beads (Beckman) were used to purify cDNA fragments, and biotin-indexes were tagged by 4 PCR cycles. cDNAs were fragmented
to around 300 bp using Covaris S2 (Covaris) and recaptured by Dynabeads MyOneTM Streptavidin C1 beads (Thermo Fisher). cDNA
library was amplified using KAPA Hyper Prep Kit and sequenced on an Illumina Hiseq X Ten platform (Novogene). |
2021-01-10 |