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| Status |
Public on Jan 30, 2019 |
| Title |
control_1 (3'SAGE-seq) |
| Sample type |
SRA |
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| Source name |
lung fibroblast
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Col1a2-GFP Transgenic
tissue: lung
cell type: lineage (CD45, CD31, EpCAM, CD146 and Ter119) negative, propidium iodide negative, Col1a2-GFP and LNGFR positive fibroblast
expression: control
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| Treatment protocol |
C57BL/6 mice were anesthetized with isoflurane before a single dose of bleomycin sulfate (2 mg/kg dissolved in 50 μL of sterile saline solution; Toronto Research Chemical, Toronto, ON, Canada) was instilled by oropharyngeal aspiration. Primary fibroblasts were isolated from Col1a2-GFP murine lungs and active form of Srebf1c (trSrebf1) and control vector (marker : hLNGFR) were retrovirally transduced. 5x10^6 of each transduced cell were intratracheally-transferred to B6 mice at day 5 post-administration of 2 mg/kg of bleomycin. Expression of trSrebf1c was induced by doxycycline administration at day 7 post-administration of 2 mg/kg of bleomycin.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Transferred lung fibroblasts were isolated from bleomycin-treated lungs of C57BL/6 mice at day 5 post-transfer. 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 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+ hLNGFR+ live fibroblasts were further purified by cell sorting using a FACS Aria II (BD).
PolyA RNAs were isolated and amplified from donor 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).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
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| Data processing |
Adapter trimming and quality filtering of sequencing data were performed by using Trimommatic-v0.36 and PRINSEQ-0.20.4. Filtered reads were mapped to Refseq mm10 by using Bowtie2-2.2.5. Reads that were not mapped to NlaIII sites were removed, and quantified tag numbers of each gene as the expression level of each gene.
Between-sample normalization was performed by using Microsoft R Open 3.3.3 and TCC package.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include raw tag-count values for each sample
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| Submission date |
Feb 13, 2018 |
| Last update date |
Jan 30, 2019 |
| Contact name |
Shigeyuki Shichino |
| E-mail(s) |
s_shichino@rs.tus.ac.jp
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| Organization name |
Tokyo University of Science
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| Department |
Department of Molecular regulation of Inflammatory and Immune Diseases
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| Street address |
2641, Yamasaki, Noda-shi
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| City |
Chiba |
| ZIP/Postal code |
278-0022 |
| Country |
Japan |
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| Platform ID |
GPL18635 |
| Series (2) |
| GSE110533 |
Transcriptomic analysis of active form of Srebf1-overexpressed fibroblasts in bleomycin-induced lung fibrosis |
| GSE110711 |
Transcriptome network analysis reveals the LXR-Srebf1 axis as protective hub of fibroblast activation in murine pulmonary fibrosis |
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| Relations |
| BioSample |
SAMN08524377 |
| SRA |
SRX3688953 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2995327_Control_1.txt.gz |
96.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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