GSM8421253: poolD-89; Mus musculus; RNA-Seq
Single cells were sorted (BD FACSAriaIII) in 96 well plates (VWR) containing 2μL of PBS including 0.2% Triton X-100 and 4U of RNase inhibitor (Takara) per well. Plates were properly sealed and spun down at 2000g for 1min before storing at −80°C. Whole transcriptome amplification was performed with a modified SMART-seq2 protocol as described previously, using 20 instead of 18 cycles of cDNA amplification. PCR purification was realized with a 0.8:1 ratio (ampureXP beads:DNA). Amplified cDNA quality was monitored with a high sensitivity DNA chip (Agilent) using the Bioanalyzer (Agilent) SMART-seq2 based scRNA-seq; Sequencing libraries were performed using the Nextera XT kit (Illumina) as described previously, using 1/4th of the recommended reagent volumes and 1/5th of input DNA with a tagmentation time of 9min. Library quality was monitored with a high sensitivity DNA chip (Agilent) using the Bioanalyzer (Agilent). Indexing was performed with the Nextera XT index Kit V2 (A-D).