title: GSM6030580: mouse_Organoid_2; 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°C, and 5 min at 72°C, followed by 5 min at 72°C, and hold at 4°C. The first PCR products were purified by AmPure XP beads (Beckman-Coulter) at a 0.6:1 ratio of reagents to sample and eluted with 25 μL of nuclease-free water. 6.3uL of purified first PCR product was mixed with the 8.7 μL of second PCR mix [0.4 μM of primers (illumina-i7, NH2-3' WTA), and 1× KAPA Hifi Hotstart ReadyMix], and the thermal cycling was performed as following condition: for 3 min at 95°C, 5 cycles of 20 sec at 98°C, 15 sec at 65°C, and 5 min at 72°C, followed by 5 min at 72°C, and hold at 4°C. The second PCR products were purified by AmPure XP beads at a 0.6:1 ratio of reagent to sample and eluted with 15 μL of 10 mM Tris-HCl (pH 8.0). 100 ng of the whole-transcriptome library was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs) with some modifications. The thermal cycling was performed as following condition: for 7 min at 32ºC, 30 min at 65ºC, and hold at 4ºC. Then, 1.25 μL of 1.5 μM illumine adapter was used for adapter ligation. Ligated products were purified by double size selection with 0.4× → 0.7× (final 1.1×) AmPure XP beads and eluted with 10 μL of nuclease-free water. The barcoding PCR was performed with 22.5 uL of barcoding mix [7.5 μL of the resulted eluates, 1 μM primers (ILMN_[UDI]_i5 and ILMN_[UDI]_i7), and 1× NEBNext Ultra II Q5 (New England Biolabs)], and the thermal cycling was performed as following condition: for 30 sec at 98°C, 9 cycles of 10 sec at 98°C and 75 sec at 65°C, followed by 5 min at 65°C, and hold at 4°C. Resultant products were purified twice by double size selection with 0.5× → 0.8 × (final 1.3×) AmPure XP beads and eluted with 12 μL of 10 mM Tris-HCl pH8.0. Size distribution of amplified products was analyzed by MultiNA system (Shimazu) with appropriate dilutions. Final transcriptome libraries, whose lengths were around 300 base pairs, were quantified using the KAPA Library Quantification Kit (KAPA Biosystems). Pooled libraries were sequenced by Illumina Novaseq 6000 sequencer (Illumina).