GSM9108411: mFetalIntestinal_WT; Mus musculus; RNA-Seq
After single cell dissociation using TrypLE at 37°C cells were filtered through a 40 µm nylon cell strainer (Falcon). Variable single cells were FACS sorted into 384-well cell capture plated (Single Cell Discoveries). Each well of a cell capture plate contains a small 50nl droplet of barcoded primers and 10µl of mineral (Signa, M8410). After sorting, plates were immediately spun and placed on dry ice. Plated were stored at -80ºC. scRNA-seq was performed by Single Cell Discoveries according to an adapted version of the SORT-seq protocol15 with primers described in earlier. Cells were heat-lysed at 65ºC followed by cDNA synthesis. After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol17 to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina). The DNA library was paired-end sequenced on an Illumina NextSeq500 at paired-end 60- and 26-bp read length and 75,000 reads per cell.