title: GSM6030588: mouse_Organoid_10; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were minced into 0.5 mm^2 with a razor blade and digested with Collagenase solution [RPMI-1640 (Nacalai Tesque) supplemented with 10% FBS (MP Bio Japan), 10 mM HEPES pH7.2-7.4 (Cytiva (Global Life Sciences Technologies Japan)), 0.2% Collagenase (Fujifilm Wako Pure Chemicals), 2 kU/mL DNase I (Merck)] , and 0.96 mg/mL Dispase (Roche) at 37 ºC for 50 minutes. Cell suspensions were washed and resuspended with 25% Percoll PLUS (Cytiva). After under layered 65% Percoll PLUS, cell suspensions were centrifuged at 20ºC for 1,000 ×g for 20 minutes, and the middle layer was collected. Resultant cell suspensions were washed and resuspended with 1000 μL of preparation medium [RPMI-1640 supplemented with 5% FBS and 10 mM HEPES]. Each single-cell suspension cell concentration was counted using Flow-count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter). Transcriptome libraries were prepared on cell lysate resolved in lysis buffer. PolyA RNAs were isolated using Dynabeads M-270 Streptavidin (Thermo Fisher Scientific) conjugated with biotin-3' WTA-EcoP-dT25 according to the previous report with some modifications (GSE110711). To perform reverse transcription, beads were then suspended in 10 μL of RT mix [5× Superscript IV buffer (Thermo Fisher Scientific), 1 mM dNTP (Roche), 5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma), 6 mM MgCl2, 1 U/μL RNaseIn Plus Rnase Inhibitor (Promega), 10 U/μL Superscript IV (Thermo Fisher Scientific)], and incubated for 5 min at 35ºC, 30 min at 42ºC, and immediately cooled on ice. Beads were washed once with B&W-T buffer [5 mM Tris-HCl (pH 8.0) (Nippon Gene), 1 M NaCl (Merck), 1 mM EDTA (Nippon Gene) and 0.05% Tween-20 (Merck)], and once with Tris-HCl (pH 8.0). To digest free-primer, beads were then suspended in 10 μL of Exo I mix [10× Exo I buffer (New England Biolabs), 2 U/μL Exonuclease I (New England Biolabs)], and incubated for 30 min at 37ºC and immediately cooled on ice. Beads were washed twice with B&W-T buffer, and once with Tris-HCl (pH 8.0). To add polyC tail, beads were then suspended in 10 μL of polyC tailing mix [10× Thermopol buffer (New England Biolabs), 2 mM dCTP (Roche), 0.1 mM ddCTP (GE Healthcare), 1 mM CoCl2 (Roche), RNaseH (Invitrogen), 15 U/ul TdT enzyme (Roche)], and incubated for 30 min at 37ºC and immediately cooled on ice. Beads were washed once with B&W-T buffer, and once with Tris-HCl (pH 8.0). To synthesis 2nd strand, beads were then suspended in 10 μL of 2nd strand mix [1x KAPA Hifi Hotstart ReadyMix (KAPA Biosystems), 0.4 μM of primer (illumina-i7-9G)], and the thermal cycling was performed as following condition: for 3 min at 95°C, 20 sec at 98°C, 16 cycles of 1 min at 47°C and 2 min at 72°C, followed by 5 min at 72°C, and hold at 4°C. To amplify the total cDNA, 10 μL of beads containing cDNA was added with 15 μL of first PCR mix [0.32 μM of primer (illumina-i7), 0.48 μM of primer (NH2-3' WTA), and 1x KAPA Hifi Hotstart ReadyMix], and the thermal cycling was performed as following condition: for 3 min at 95°C, 12 cycles of 20 sec at 98°C, 15 sec at 65°