title: GSM8059075: Oesophagus, replicate1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: C57Bl/6 mice were i.p injected with 100 𝜇L pentobarbital and perfused with at least 50 mL ice-cold DPBS. The oesophagus was dissected and the muscularis externa mechanically removed. The oesophagus was longitudinally opened using spring-scissors with a ball tip and incubated in 0.5 mg/mL Thermolysin for 15 min at 37°C. Subsequently, stroma and epithelium were separated using forceps under a dissection microscope and cut into 3 equally sized tissue pieces. Afterwards, tissue samples were thoroughly minced and incubated in 0.25 mg/mL Liberase™ and 0.25 mg/mL DNase I in HBSS at 37°C for 30 min or 60 min for stroma and epithelium, respectively. Samples were thoroughly pipetted up and down every 15 min using a 200 𝜇L pipette tip. In addition, epithelial samples were run on a gentleMACS™ Dissociator. Cells ere washed twice in 1% FBS/PBS and kept on ice until sorting using a BD FACSMelody™ instrument. Library preparation was performed according to Hagemann-Jensen, Michael, et al. "Single-cell RNA counting at allele and isoform resolution using Smart-seq3." Nature Biotechnology 38.6 (2020): 708-714. In short, cells were sorted and lysed in 384- well plates. Afterwards, 10 cycles of reverse transcription, 22 cycles of cDNA amplification, cDNA purification, normalisation and dilution to 100 pg/ml was performed. Subsequently, tagmentation and 12 cycles of index PCR using Nextera Index primers were done. Subsequently, cDNA libraries were pooled and purified. To prepare sequencing on an MGI platform, circular ssDNA libraries were created and adapter conversion PCR run for 5 cycles followed by circularisation. DNA nanoballs were created and 100 bp paired-end sequencing was run.