GSM5348517: 13T_Super-engaged 1; Homo sapiens; RNA-Seq
TEGs (>0,8x106 per condition) were  co-cultured with 13T PDOs (E:T of 1: 3) and separated at 4 hrs into organoid-engaged and organoid non-engaged populations by a slow-spin (30 rcf) centrifugation step, co-cultured for another 2h with or without addition of fresh PDOs, again followed 2 slow-spin (30 rcf) centrifugation steps to obtain non-engagedEnriched and super-engaged TEG populations. To create single-cell suspensions, conditions containing organoids (all 'engaged' TEG conditions) were treated with TrypLE for seven minutes at 37°C and washed with adDMEM/F12+++. Cells were then stained in FC buffer (2% FCS in PBS) with anti-CD3-APC conjugated antibodies (1:80; BioLegend) and LIVE/DEAD Fixable Near-IR Dead Cell Stain (1:1000; ThermoFisher) for 30 minutes at 4°C and sorted into 384-wells SORTseq plates using a FACS Aria Cell Sorter (BD Biosciences) and directly stored at -80°C until further processing. All sorted plates were processed according to the CEL-Seq2 protocol with the total transcriptome amplification via poly-A RNA-capture, library preparation, and sequencing into Illumina sequencing libraries as previously described(Muraro et al., 2016). Paired-end sequencing (read1: 30 bp; read2: 120 bp) was used to sequence the prepared libraries using an Illumina NextSeq sequencer.