GSM5233646: 5pcO2_r1_snRNA-seq; Homo sapiens; OTHER
On day 8 of differentiation (cells cultured in BAP/DMEM/F12/KOSR for 3 days and AP/DMEM/F12/KOSR until day 8), each culture well was rinsed twice with DMEM/F12 (Thermo Scientific)  and the colonies partially dispersed by using gentle cell disassociation reagent (GDR) (Stemcell Technologies) (0.6 ml/well; 7 minutes (min)) at 37 °C. The colonies were fully dislodged from the substratum by gentle scraping and nuclei were isolated from the disassociated cells using the protocol provided by 10X genomics with minor modification. Briefly, 1 ml of chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% Nonidet™ P40 Substitute in Nuclease-Free Water) was added to each well through a wide-bore pipet tip. After gently pipetting several times, the suspension was kept on ice for 5 min to achieve maximum lysis of cells and then centrifuged (500x g; 5 min at 4°C). The pellet was resuspended in 1 ml of nuclear wash and resuspension buffer (NWRB) (1X PBS with 1.0 % w/v bovine serum albumin) and 0.2U/μl RNase inhibitor (Roche, cat# 03335399001), cell debris was removed by filtration through a 40 µm Nylon cell strainer (Falcon), and recentrifuged as above. The supernatant solution was removed, and the final nuclear pellet suspended in 1ml of NWRB. Libraries were constructed by following the manufacturer's (10x Genomics) protocol with reagents supplied in 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1.  Briefly, nuclear suspension concentration and viability (unbroken cells) were measured with an Invitrogen Countess II automated cell counter. Nuclear suspension (1000 nuclei per microliter), reverse transcription master mix, and partitioning oil were loaded on a Chromium Next GEM G chip with a nuclear capture target of 5,000 nuclei per library.  Post-Chromium controller GEMs were transferred to a PCR strip tube and reverse transcription performed on an Applied Biosystems Veriti thermal cycler at 53°C for 45 min.  cDNA was amplified for 14 cycles and purified using Axygen AxyPrep MagPCR Clean-up beads.  cDNA fragmentation, end-repair, A-tailing and ligation of sequencing adaptors was performed according to manufacturer specifications.  The final library was quantified with the Qubit HS DNA kit and the fragment size was analyzed using an Agilent Fragment Analyzer system.  Libraries were pooled and sequenced on an Illumina NovaSeq S1 flow cell with a goal to generate 50,000 reads per nucleus with a sequencing configuration of 28 base pair (bp) on read1 and 91 bp on read2.