 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 08, 2021 |
| Title |
PROseq_TGFb_siCtrl_Set2 |
| Sample type |
SRA |
| |
|
| Source name |
Cardiac fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Cardiac fibroblasts
treatment: TGFb
type of experiment: PRO seq
|
| Growth protocol |
A Tcf21MCM mouse was crossed with a Rosa26-Ai6 mouse (Jackson Laboratory stock# 007906). At 10 weeks of age, intraperitoneal injection of Tamoxifen (75 mg tamoxifen/kg) was done for 5 days (once a day). Tamoxifen was prepared following Jackson Laboratory guidelines https://www.jax.org/research-and-faculty/resources/cre-repository/tamoxifen#). After 5 days of injection, the mouse was sacrificed and the non-CM cells isolated as described in a previous section of this method. 100k ZsGreen positive cells were sorted with BD AriaII sorter and cultured for 3 days at 37C in a humidified incubator with 5% CO2 and maintained in FB medium: high glucose DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Hyclone, GE Healthcare), 1x Non-Essential Amino Acid (NEAA), 10U/ml penicillin/streptomycin and 1mM sodium pyruvate (all from Life Technologies). For immortalization, FB were seeded at 105 per 100 mm plate in the afternoon. Next morning, 2 hours before infection, fresh media was added on plates. The SV40 T Antigen Cell Immortalization Kit (Alstem, #CILV01) was used. 5 µl/well lentivirus supernatant (vials have 20ul) in the presence of Polybrene (added to a final concentration of 5 µg/ml). The next day, remove viral supernatant and switch again to FM medium. After 72 hours incubation, subculture the cells into 2 × 100 mm dishes and add the appropriate amount of puromycin for stable cell-line generation (1ug/ml for mouse cardiac FBs).
After 10 days after puromycin selection, multiple clones were picked for expansion. For daily cell culture, FBs were split every 3-4 days and the media was changed every other day. For the FB TGF-β stimulation, FBs were seeded at 1x105/well of 6 well plate at day 1. On day 2, the media was changed to same basal media with 0.5% FBS. On the day 3, TGF-1(Peprotech) was added into the media at concentration of 10ng/ml. Cells were collected on day 5 for RNA extraction and subsequent gene expression analysis with real time PCR.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 48h of TGF-b treatment, fibroblast cells were washed 3 times with cold PBS and then sequentially swelled in swelling buffer (10mM Tris-HCl pH7.5, 2mM MgCl2, 3mM CaCl2) for 10 min on ice, harvested, and lysed in lysis buffer (swelling buffer plus 0.5% NP-40, 20 units of SUPERase-In, and 10% glycerol). The resultant nuclei were washed two more times with 5ml lysis buffer and finally resuspended in 100 μl of freezing buffer (50mM Tris-HCl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For the run-on assay, resuspended nuclei were mixed with an equal volume of reaction buffer (10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 20 units of SUPERase-In, 1% sarkosyl, 100 M A/GTP, 100 μM biotin-11-C/UTP (Perkin-Elmer) and incubated for 5 min at 30°C. The resultant nuclear-runon RNA (NRO-RNA) was then extracted with TRIzol® LS reagent (Life Technologies, Cat# 10296–028) following manufacturer’s instructions. NRO-RNA was fragmented to 200–500nt by alkaline base hydrolysis on ice for 30 min and neutralized by adding 1× volume of 1 M Tris-HCl pH 6.8, Excessive salt and residual NTPs were removed by using P-30 column (Bio-Rad, Cat# 732–6250), followed by treatment with DNase I (Promega Cat# M6101) and antarctic phosphatase (NEB Cat# M0289L). Fragmented nascent RNA was bound to 10 μl of MyOne Streptavidin C1 dynabeads (Invitrogen, Cat# 65001) following the manufacturer’s instructions. The beads were washed twice in high salt (2 M NaCl, 50 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 0.5 mM EDTA), once in medium salt (1M NaCl, 5 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 0.5 mM EDTA), and once in low salt (5 mM Tris-HCl pH 7.5, 0.1% Triton X-100). Bound RNA was extracted from the bead using Trizol (Invitrogen, Cat# 15596–018) in two consecutive extractions, and the RNA fractions were pooled, followed by ethanol precipitation.
Libraries were generated using the NEBNext® Multiplex Small RNA Library Prep Set. (NEB, Cat#E7300S) following the manufacturer’s instructions. The cDNA products were separated on a 10% polyacrylamide TBE-urea gel and only those fragments migrating between 200–500bp were excised and recovered by gel extraction. Finally, libraries were quantified by Qubit and sent to sequence SR75 bp on a HiSeq 4000 platform (Illumina).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Cluster generation, sequencing, and base calling was performed on Illumina HiSeq 4000
Sequenced reads were trimmed for adaptor sequence and low-quality reads removed using Trimmomatic.
Reads were mapped to the mm10 genome with Bowtie 2. All the downstream analysis was done using Homer. Reads Per Kilobase of exon per Megabase of library size (RPKM) for every gene were calculated to quantify nascent transcription.
Library strategy: PROseq
Genome_build: Mm10
Supplementary_files_format_and_content: We provide BigWig files for PROseq We provide bed files and bigWig files for ChIPseq.
|
| |
|
| Submission date |
Dec 28, 2020 |
| Last update date |
Apr 08, 2021 |
| Contact name |
Pawel Przytycki |
| E-mail(s) |
pawel.przytycki@gladstone.ucsf.edu
|
| Organization name |
Gladstone Institutes
|
| Department |
GIDB
|
| Lab |
Katie Pollard
|
| Street address |
1650 Owens Street
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE155882 |
A Transcriptional Switch Governs Fibroblast Activation in Heart Disease |
|
| Relations |
| BioSample |
SAMN17170348 |
| SRA |
SRX9740996 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4990674_PROseq_aCFs_set2_siCTRL.mTDneg.ucsc.bigWig |
144.9 Mb |
(ftp)(http) |
BIGWIG |
| GSM4990674_PROseq_aCFs_set2_siCTRL.mTDpos.ucsc.bigWig |
137.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
|
|
|
|
 |
