GSM2872695: RPE_8W_emb3_rep1; Homo sapiens; RNA-Seq
The modified Smart-seq2 protocol for multiplexed single-cell RNA-seq was described previously. Briefly, after digestion, a single retinal cells was randomly picked into 2μL cell lysis buffer using a mouth pipette. The reverse transcription reaction was performed with 24 oligo (dT) primer anchored with the 8 bp cell specific barcode, and also with 8 bp unique molecular identifiers (UMIs). After the first-strand reaction, the cDNA was amplified by 19 cycles. Then the amplified cDNA were pooled together to proceed the following steps. Biotinylated pre-indexed primer was used to amplify the PCR product by additional 4 cycles to introduce biotin tags to the 3' end of the amplified cDNA. About 300 ng DNA was sheared to around 300 bp by Covaris and the 3' ternimal of the DNA was captured by Dynabeads® MyOne™ Streptavidin C1 beads. RNA-seq library was constructed using Kapa Hyper Prep Kit and subjected to 150 bp paired-end sequencing on illumina HiSeq 4000 platform.