title: GSM4614440: ESC RNA rep1; Homo sapiens; RNA-Seq
library_construction_protocol: We purified mRNA from the RNA using the Oligotex mRNA prep kit (Qiagen) and synthesized cDNA from the resulting mRNA with SuperScript II RT (Invitrogen), using a barcode specific primer with a unique molecular identifier (UMI) (P7-pLSmp-ass16UMI-gfp) DNA fragments containing barcodes were amplified from both the isolated DNA and generated cDNA from each replicate of each cell type with 3-cycle PCR using primers targeting the barcoded region (P7-pLSmp-ass16UMI-gfp and P5-pLSmP-5bc-i#). These primers also contained a sample index for demultiplexing and a UMI for consolidating replicate molecules (see later). A second round of PCR was performed to attach the adapters necessary for sequencing (P5, P7). The fragments were purified and further sequenced with six runs of NextSeq 15PE with 10-cycle dual index reads, using custom primers (R1, pLSmP-ass-seq-ind1; R2 (read for UMI), pLSmP-UMI-seq; R3, pLSmP-bc-seq; R4 (read for sample index), pLSmP-5bc-seq-R2).
identifiers.namespace: GEO
identifiers.id: GSM4614440