GSM1625895: Control Library; Mus musculus; RNA-Seq
Organoids were dissociated into single-cell suspension using TrypleE enzyme mix. The cells were pelleted and resuspended in F12 medium with 5 to 10% serum. Single cells were FACS-sorted into 96-well plates, containing 100ml Trizol reagent. X of Spike-in RNA was added to each well. Total RNA was extracted from each cell, according to the Trizol manufacturer protocol. Each single-cell RNA sample was processed for CEL-Seq. Single cells were processed using the previously described CEL-seq technique, with a few alterations. A 4bp random barcode as unique molecular identifier (UMI) was added to the primer in between the cell specific barcode and the poly T stretch. Instead of the 70° C lysis step, cells were lysed by adding 0.05% IGEPAL CA 630 (sigma) to the first strand synthesis mix. Libraries were sequenced on an Illumina HighSeq 2500 using 50bp paired end sequencing.