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| Status |
Public on Oct 08, 2019 |
| Title |
BMDMctrTgfb_rep2 |
| Sample type |
SRA |
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| Source name |
Smad4 flox/flox
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| Organism |
Mus musculus |
| Characteristics |
strain: Smad4 flox/flox
treatment1: in vitro
treatment2: 4 day cultured BMDMs
treatment3: TGF-beta 24h
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from in vitro culture cells and purified using a Direct-zol RNA MicroPrep Kit (Zymo Research) according to the manufacturer’s instructions (Zymo Research). FACS purified cells were pelleted and put into 150 μl lysis/Oligo d(T) Magnetic Beads binding buffer (100mM Tris-HCl pH7.5, 500mM LiCl, 10mM EDTA pH8.0, 1% LiDS, 5mM DTT) and stored at –80°C until processing. mRNAs were enriched by incubation with Oligo d(T) Magnetic Beads (NEB, S1419S).
Poly A enriched mRNA was fragmented, in 2x Superscript III first-strand buffer with 10mM DTT (Thermo Fisher Scientific), by incubation at 94°C for 9 minutes, then immediately chilled on ice before the next step. The 10 µL of fragmented mRNA, 0.5 µL of Random primers (3 µg/µL) (Thermo Fisher Scientific), 0.5 µL of Oligo dT primer (50 µM) (Thermo Fisher Scientific), 0.5 µL of SUPERase-In (Ambion), 1 µL of dNTPs (10 mM) and 1 µL of DTT (10 mM) were heated at 50°C for one minute. At the end of incubation, 5.8 µL of water, 1 µL of DTT (100 mM), 0.1 µL Actinomycin D (2 µg/µL), 0.2 µL of 1% Tween-20 (Sigma) and 0.5 µL of Superscript III (Thermo Fisher Scientific) were added and incubated in a PCR machine using the following conditions: 25°C for 10 minutes, 50°C for 50 minutes, and a 4°C hold. The product was then purified with RNAClean XP beads (Beckman Coulter) according to manufacturer's instruction and eluted with 10 µL nuclease-free water. The RNA/cDNA double-stranded hybrid was then added to 1.5 µL of Blue Buffer (Enzymatics), 1.1 µL of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 µL of RNAse H (5 U/µL), 1.05 µL of water, 1 µL of DNA polymerase I (Enzymatics) and 0.15 µL of 1% Tween-20. The mixture was incubated at 16°C for 2.5 hours. The resulting dUTP-marked dsDNA was purified using 28 µL of SpeedBeads (GE Healthcare), diluted with 20% PEG8000, 2.5M NaCl to final of 13% PEG, eluted with 40 µL EB buffer (10 mM Tris-Cl, pH 8.5) and frozen at -80°C. The purified dsDNA (40 µL) underwent end repair by blunting, A-tailing and adapter ligation using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 16 cycles, size selected by gel extraction.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
F5.TPM.txt
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| Data processing |
Preprocessing: FASTQ files from sequencing experiments were mapped to the mouse mm10 genome. STAR with default parameters was used to map RNA-seq experiments (Dobin et al., 2013). HOMER was used to convert aligned reads into “tag directories” for further analysis (Heinz et al., 2010).
Each experiment was quantified using the “analyzeRepeats” script of HOMER. To generate a table of TPM values, the parameters -condenseGenes -count exons -tpm were used which extract only one transcript (most aboundace) for a gene.
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited text file: Table of TPM values for RNA-Seq
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| Submission date |
Mar 21, 2019 |
| Last update date |
Oct 08, 2019 |
| Contact name |
Christopher K Glass |
| E-mail(s) |
ckg@ucsd.edu
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| Organization name |
University of California, San Diego
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| Department |
School of Medicine
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE128657 |
Liver-derived signals sequentially reprogram myeloid enhancers to initiate and maintain Kupffer cell identity (RNA-Seq) |
| GSE128662 |
Liver-derived signals sequentially reprogram myeloid enhancers to initiate and maintain Kupffer cell identity |
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| Relations |
| BioSample |
SAMN11190950 |
| SRA |
SRX5550957 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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