GSM9216111: lung alveolar organoid, Capn1 knockout, plate2-2 well D02; Homo sapiens; Mus musculus; RNA-Seq
Libraries were prepared from extracted RNA using a modified Smart-seq3 protocol ( Hagemann-Jensen, M., Ziegenhain, C., Chen, P. et al. Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 38, 708–714 (2020). https://doi.org/10.1038/s41587-020-0497-0) Annealing reaction was performed with 4ul purified RNA, 0.8 ul 40% PEG 8000, 0.08 ul RNAse Inhibitor, 0.04 ul Smart-seq3 oligo dT (100µM), 0.3 ul dNTP (25mM each) and 1 ul water per 384 well reaction. 10 ul mineral oil was added on top to prevent evaporation. Reaction was sealed, spun and incubated at 70˚C for 10 min. Reverse translation was performed by adding 2 ul of RT mix (0.2ul Tris HCl, pH8.0 (1M), 0.24 ul NaCl (1M), 0.2 ul MgCl2 (100mM), 0.08 ul GTP (10mM), 0.64 ul DTT (100mM), 0.16 ul TSO (100µM), 0.08 ul Maxima H Minus RTase and 0.4 ul water) to each 384 well reaction. The RT reaction was incubated at 42°C for 90 min and repeated the following steps for 10 cycles: 50˚C for 2 min and 42˚C for 2 min, and was terminated at 85°C for 5 min. cDNA amplification was performed by adding 12ul of PCR mix (0.1 ul cDNA fwd (100µM), 0.02 ul cDNA rev (100µM), 0.1 ul MgCl2 (100mM), 10 ul 2X Kapa HiFi Hotstart Readymix  and 1.78 ul water) to the RT reaction for a total of 20 ul. Reaction was thermal cycled at 95°C for 3 min, [98°C for 20 sec, 65°C for 30 sec, 72°C for 4 min] x 14 cycles, 72 °C 4min. cDNA was diluted 1:10 in water. 1 ul of each sample was transferred to a new 384 well plate to perform tagmentation. 3 ul of tagmentation mix (0.8 ul 5X TAPS Mg buffer, 0.1 ul Tn5 enzyme, 2.1 ul water) was added to each sample on ice. Plate was sealed, vortexed for 15 seconds and spun. The reaction mixture was incubated at 55 °C for 10 min. Reaction was quenched by adding 0.5µl of 0.2% SDS to each well, sealing, vortexing for 15 seconds, and spinning the plate. The plate was  incubated at room temperature for 5 min. Libraries were indexed by added 3.5 ul of PCR mix and 2 ul indexing primers  to each sample.  Reaction was thermal cycled at 72°C for 5 min, 95°C for 3 min, [98°C for 20 sec, 65°C for 30 sec, 72°C for 30 sec] x 10 cycles, 72 °C 4min. Libraries were pooled and sequenced on an Illumina NovaSeq X Plus system, targeting 8M reads per library.
GEO
GSM9216111_r1