GSM1684386: 5 day YFP positive cells Replicate 3; 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. Primary cells were harvested from freshly isolated small intestines. Crypts were released using PBS0/EDTA followed by Trypsin dissociation. Single cells were FACS-sorted into 96-well plates, containing 100ul Trizol reagent and 0.03ul of 1:50.000 diluted Spike-In RNA. Total RNA was isolated according to the manufacturer's instructions, with the following alterations. RNA was precipitated overnight at -20 °C, with 2ug glycogen. 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. RNA pellets were dissolved in primer mix and incubated for 2 minutes at 70 °C. Libraries were sequenced on an Illumina HighSeq 2500 using 50bp paired end sequencing or Illumina NextSeq 500 using 75bp paired end sequencing.
GEO Accession
GSM1684386