GSM9313310: DFCI112_scRNA; Homo sapiens; RNA-Seq
Following sample preparation, libraries are quantified using PicoGreen. Based on PicoGreen quantification, libraries are normalized to equal concentration and pooled by equal volume. Library pools are then quantified using a Sybr Green-based qPCR assay, with PCR primers complementary to the ends of the adapters (kit purchased from Kapa Biosciences). After qPCR quantification, library pools are normalized to 2 nmol/L, denatured using 0.2 N NaOH, and diluted to 20 pmol/L, the working concentration for downstream cluster amplification and sequencing. Denatured library pools are spread across the number of sequencing lanes required to hit target coverage for all samples. Libraries are constructed using the protocol described in Fisher et al. with several modifications: first, initial genomic DNA input into shearing has been reduced from 3µg to 100ng in 50µL of solution. Second, for adapter ligation, Illumina paired end adapters have been replaced with palindromic forked adapters with unique 8 base index sequences embedded within the adapter. These index sequences enable pooling of libraries prior to sequencing. Third, custom sample preparation kits from Kapa Biosciences are now used for all enzymatic steps of the library construction process.