Instrument: Ion Torrent Proton
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Murine lung fibroblasts were isolated at 0, 7, 14, and 63 days post-induction in both models. Lungs were cut into small pieces with a razor blade and digested enzymatically using 0.2% collagenase (Wako, Osaka, Japan), 0.96 mg/mL Dispase II (Roche, Basel, Switzerland), and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) for 60 min at 37°C. Erythrocytes and silica particles were removed by 70% Percoll (GE Healthcare, Buckinghamshire, UK) density separation. The single-cell suspensions were then depleted of lineage+ (CD31+, CD45+, EpCAM+, CD146+ and Ter119+) cells by negative selection with an AutoMACS cell separator (Miltenyi Biotech, Bergisch Gladbach, Germany). Propidium iodide− lineage− Col1a2GFP+ live fibroblasts were further purified by cell sorting using a FACS Aria II (BD). PolyA RNAs were isolated and amplified from sorted fibroblasts according to the protocol described in (Huang H, Nucleic Actd Res, 2014) with some modifications. Briefly, 0.5 pmol of biotin-TEG-adapter-dT25 primers was bound to 20 μL of Dynabeads M270 streptavidin (Thermo Fisher Scientific). The washed beads (20 uL) were added to each cell lysis buffer containing 10,000 sorted fibroblasts and incubated for 30 min at room temperature with gentle rotation. Beads were washed once with wash buffer A [0.1% LiDS, 10 mM Tris-HCl (pH 7.5), 150 mM LiCl, and 1 mM EDTA] and three times with wash buffer B [10 mM Tris-HCl (pH 7.5), 150 mM LiCl, and 1 mM EDTA]. Beads were then suspended in 10 μL of RT mix 1 [1× SSIV buffer (Thermo Fisher Scientific), 2 mM dNTP, 2 M betaine (Sigma-Aldrich), 12 mM MgCl2, and 3.2 U/μL RNaseIn Plus (Promega)] and incubated for 90 s at 70ºC, 5 min at 35ºC, and immediately cooled on ice. RT mix 2 [10 μL; 1× SSIV buffer, 10 mM DTT (Thermo Fisher Scientific), 10 U/μL Superscript IV (Thermo Fisher Scientific), and 2 M betaine (Sigma-Aldrich)] was added, and reverse transcription was performed for 5 min at 35ºC and 15 min at 50ºC. Beads were washed once with cell lysis buffer, twice with B&W-T buffer [5 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.5 mM EDTA, and 0.1% Tween-20], once with Tris-HCl (pH 8.0) and 20 μL of RNase H mix [1× first-strand buffer (Life Technologies, Carlsbad, CA, USA), 5 mM DTT, 0.6 U RNase H (Thermo Fisher Scientific)], and incubated for 20 min at 37ºC to digest reverse-transcribed mRNA. Beads were washed, 20 μL of TdT mix [50 mM Tris-HCl (pH 8.0), 100 mM KCl, 3 mM MgCl2, 1 mM CoCl2 (Roche), 0.65 mM dATP (Thermo Fisher Scientific), and 15.2 U/μL TdT (Roche)] was added on an ice-chilled aluminum rack, and polyA-tailing was performed for 3 min at 37ºC. The reaction was stopped by adding 5 μL of 0.5 M EDTA, and the enzyme was heat-inactivated by incubation for 10 min at 65ºC. Beads were washed, 20 μL of second-strand synthesis mix [1× KAPA Hifi ReadyMix (KAPA Biosystems, Wilmington, MA, USA) and 0.4 µM LNA anchored tagging primer] was added, and second-strand synthesis was performed according to the following program: 95ºC for 2 min, 98ºC for 20 s, 50ºC for 2 min, 72ºC for 7 min, and hold at 4ºC. Beads were washed, and 1/4 of the beads were used for the first round of whole-transcript amplification (WTA) in 25 μL of first-round WTA mix [1× KAPA Hifi ReadyMix (KAPA Biosystems), 0.4 µM anchored tagging primer, and 0.4 µM 3′ WTA primer] using the following program: 95ºC for 3 min, five cycles of 98ºC for 20 s, 65ºC for 15 s, and 72ºC for 7 min, followed by 72ºC for 5 min and a hold at 4ºC. PCR products were purified twice with 0.6× AmPure XP beads (Beckman Coulter) and eluted with 23 μL of nuclease-free water. Second-round WTA mix [27 μL; 1× KAPA Hifi ReadyMix (KAPA Biosystems), 0.614 µM 5′ WTA primer, and 0.614 µM Biotin-TEG-3′ WTA primer] was added, and the second round of WTA was performed using the following program: 95ºC for 3 min, nine cycles of 98ºC for 20 s, 65ºC for 15 s, and 72ºC for 7 min, followed by 72ºC for 5 min and a hold at 4ºC. PCR products were purified twice with 0.6× AmPure XP beads and eluted with 25 μL of Tris-HCl (pH 8.0). Amplified whole transcripts were quantified using a Nanodrop 1000 (Thermo Fisher Scientific), and size distribution was analyzed by agarose electrophoresis and SYBR Gold staining (Thermo Fisher Scientific). 3'SAGE-seq libraries were constructed according to the protocol described in (Matsumura H, Methods Mol Biol, 2012) with some modifications. Briefly, 150 ng of the whole-transcript library was restriction digested with NlaIII (New England Biolabs, Ipswich, MA, USA) for 2 h at 37ºC, and biotinylated 3′-tail transcripts were immobilized on Dynabead M-280 streptavidin beads (Thermo Fisher Scientific). Beads were washed, and 10 pmol of the CS1-EcoP15I-NlaIII adapter was ligated using a DNA ligation kit (Mighty Mix; Takara) for 30 min at 16ºC. Beads were washed three times with B&W-T buffer, once with 10 mM Tris-HCl (pH 8.0), suspended in 200 μL of EcoP15I digestion mix [1× NEBuffer 3.1, 1 mM ATP, and 0.2 U EcoP15I (New England Biolabs)], and digested for 16 h at 37ºC in a tightly sealed screw-cap tube with gentle rotation. Supernatant was purified using a Nucleospin Gel&PCR clean-up kit (Takara) and eluted twice with 12.5 μL of nuclease-free water. End-repair/polyA-tailing/ligation reactions were performed using NEBNext Ultra II modules (New England Biolabs) and 1.875 pmol of CS2-adapter according to manufacturer instructions. Reaction products were purified using a Qiagen MinElute Column (Qiagen, Hilden, Germany) and eluted with 13 μL of nuclease-free water. Barcoding mix [14.25 μL; 1× KAPA Hifi ReadyMix (KAPA Biosystems), 0.614 µM IonA-BC[N]-CS1-primer, and 0.614 µM Ion-trP1-CS2 primer] was added into 10.75 μL of elutant, and PCR enrichment was performed using the following program: 98ºC for 45 s, nine cycles of 98ºC for 15 s, 65ºC for 30 s, and 72ºC for 90 s, followed by 72ºC for 1 min and a hold at 4ºC. Reaction products were purified using double-size selection of AmPure XP beads (0.8× → 0.8×) and eluted with 10 μL of Tris-HCl (pH 8.0). The size distribution of each library was analyzed using an Agilent DNA high-sensitivity kit (Agilent Technologies, Santa Clara, CA, USA), and library concentration was quantified using a KAPA library quantification kit for Ion Torrent (KAPA Biosystems). Sequencing was performed by using Ion Hi-Q Chef Kit, Ion PI v3 Chip Kit and Ion Proton Sequencer (Thermo Fisher Scientific) according to the manufacture's instructions except the input library concentration (100 pM) and cycle number (200).