GSM7908791: hNMSO_d75_snRNA; Homo sapiens; RNA-Seq
For single-nucleus sequencing, organoids of the same stage were pooled together and were frozen immediately at - 80℃.  The Chromium Nuclei Isolation Kit with RNase Inhibitor was used according to the manufacturer's instructions to isolate nuclei for subsequent sequencing steps. In short, frozen organoids were homogenized with a pestle in the Lysis Buffer and passed through a column. Next, the debris was removed via centrifugation in the Debris Removal Buffer. For single-nucleus sequencing, nuclei were used to generate cDNA libraries using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index). Briefly, cell nuclei were partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs), and microfluidic cell nuclei were streamed at limiting dilution into a stream of Single Cell 3' v3.1 Gel Beads, and then a stream of oil. After lysis, an Illumina TruSeq Read 1, a 16 base pair 10x Barcode, a 12 bp unique molecular identifier (UMI), a 30 nt poly(dT) sequence, and a Capture Sequence 1 were released and mixed with the lysate and the Master Mix containing the RT reagents, full-length cDNA from poly-adenylated messenger RNA was generated. After individual droplets were broken down, homogenized, and cleaned from the remaining noncDNA with silane magnetic beads, the libraries were size-selected, followed by the addition of TruSeq Read 2, P5, P7, i7, and i5 sample indexes sequences to each selected cDNA. Finally, each library was sequenced using the Illumina Novaseq6000, read1 28bp, read2 151bp Mode.