title: GSM3018455: Pooled single cell RNA-Seq; Mus musculus; RNA-Seq
library_construction_protocol: Viable single cells from each treatment group were sorted based on GR1+/- phenotype on the BD FACSAria III, directly into pre-prepared 384 well plates that contained a mixture of oligo (dT) primer, dNTPs and ERCC spike ins, as outlined in the Cel-seq2 protocol. A modified version of Cel-seq2 was used to generate single cell cDNA libraries. Briefly, the sorted cells were lysed and the primers annealed to the RNA at 65°C. Primers contained a 24bp polyT stretch, a 6bp unique molecular identifier (UMI), a cell-specific 8bp barcode, the 5′ Illumina adaptor and a T7 promoter. Reverse transcription, second strand synthesis and in vitro transcription were performed according to the Cel-seq2 protocol, with the exception of the cDNA cleanup step prior to IVT, whereby the volume of the Agencourt AMPure XP beads (A63880, Beckman Coulter) was 5% of the total volume of beads used in the original protocol. Following IVT, the amplified RNA was fragmented, cleaned up and reverse transcribed with a random hexamer primer containing the sequence complimentary to the 3' Illumina adaptor. Fragments containing both adaptors were selected for by PCR using TruSeq Small RNA primers (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM3018455