GSM6253591: Experimental; Mus musculus; RNA-Seq
Single cell suspension of BM-derived cells in RT Master Mix (75 µl) was loaded on the Single Cell A Chip and partitioned with a pool of ~750,000 barcoded gel beads to form nanoliter-scale Gel Beads-In-Emulsions (GEMs). Each gel bead has primers containing: (i) an Illumina R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence. Upon dissolution of the Gel Beads in a GEM, the primers were released and mixed and incubated with cell lysate and RT Master Mix, producing barcoded, full-length cDNA from poly-adenylated mRNA. Silane magnetic beads were used to isolate cDNA from leftover biochemical reagents and primers in the post GEM reaction mixture. Full-length, barcoded cDNA was then amplified by PCR to generate sufficient DNA for library construction. Enzymatic fragmentation and size selection were used to optimize cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) were added during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via end repair, A-tailing, adaptor ligation and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.