title: GSM5469265: EC_Sham_scDrop_seq_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Individual cells were then packaged in picoliter droplets together with the barcoded 'Seq B' microparticles (Chemgenes Corp; Wilmington, MA; referenced in the Macosko et al. (PMID 26000488)) following drop-seq laboratory protocol v. 3.1 from McCarrol Lab (http://mccarrolllab.org/dropseq/; accessed last time March 3rd, 2021). For packaging, the high-throughput single-cell RNA sequencing system from Dolomite Bio (Royston, UK) was used. In brief, the beads were resuspended in lysis buffer at a concentration of 600 beads/µl and then loaded into a sample loop. The cells were resuspended at a concentration of 300 cells/µl in PBS, including 0.01% bovine serum albumin (BSA), and then loaded into an agitated tube. Monodisperse droplets were generated in droplet generation oil (Bio-Rad) by pumping the suspensions through glass single cell RNA seq chips (Dolomite Bio) at a speed of 200 µl/min for the oil and 30 µl/min for beads and cells. The aqueous droplets containing single cells and/or beads in oil were collected in 50 ml falcon tubes. We immediately proceeded with droplet breakage and cDNA synthesis according to the drop-seq laboratory protocol v. 3.1 from McCarrol Lab. Briefly, droplet contents (mRNA bound on the beads' oligos) were released and washed from oil using 6x SSC buffer and perfluorooctanol, and cDNA was generated by reverse transcription using Maxima H-RTase (Thermofisher Scientific). Subsequently, beads were subjected to exonuclease I treatment prior cDNA amplification by SMART PCR (4 + 14-18 cycles), purification, and tagmentation for sequencing exactly according to the drop-seq laboratory protocol v. 3.1. BGI