GSM3936989: Drop_seq_P12; Mus musculus; RNA-Seq
Tumor tissues were digested by TrypLE added with collagenase type I, and single cell suspension was treated by Red Blood Cell Lysis Buffer, the sequencing followed Drop-seq protocols. Briefly, monodisperse droplets ∼1 nl in size were generated using the microfluidic device, in which barcoded microparticles, suspended in lysis buffer. As soon as droplet generation was complete, droplets were broken with perfluorooctanol in 30 ml of 6× SSC. The addition of a large aqueous volume to the droplets reduces hybridization events after droplet breakage. The beads were then washed and resuspended in a reverse transcriptase mix, followed by a treatment with exonuclease I to remove unextended primers, and PCR amplified. The amplified cDNA was quantified on a BioAnalyzer High Sensitivity Chip (Agilent). cDNA size selection was performed to reduce reads that end up just giving PolyA tail sequences. The cDNA was then fragmented and amplified for sequencing with the Nextera XT DNA sample prep kit (Illumina) using custom primers that enabled the specific amplification of only the 3' ends. The libraries were purified, quantified, and then sequenced on the Illumina Hiseq 2500.
GEO Accession
GSM3936989