GSM6939551: NSC_009; Mus musculus; RNA-Seq
Viable single cells were FACS sorted into 384-well plates (Single Cell Discoveries). Each well of a cell capture plate contains a 50 nl droplet of barcoded primers and 10 µl of mineral oil (Sigma M8410). After sorting, plates were immediately spun and placed on dry ice. Plates were stored at -80°C and shipped to Single Cell Discoveries on dry ice, where single-cell RNA sequencing library prep was performed. Cells were heat-lysed at 65°C. Libraries for each plate were prepared according to an adapted version of the SORT-seq protocol (Muraro et al. 2016 with primers described in van den Brink et al. 2017). Briefly, after cell lysis, second-strand cDNA synthesis was done, and 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 protocol (Hashimshony et al. 2016)  to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina). The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1×75 bp Illumina kit (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles)