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| Status |
Public on Aug 08, 2018 |
| Title |
Gli2+RSmoY_2896 |
| Sample type |
SRA |
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| Source name |
Lung stroma
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| Organism |
Mus musculus |
| Characteristics |
tissue: lung
developmental stage: adult
treatment: administered intraperitoneally with tamoxifen for labelling the Hh-activated GLI2+ cells
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| Treatment protocol |
Tamoxifen (Sigma) was dissolved in corn oil and administered intraperitoneally at 200mg/kg per day x 5 days for activating hedgehog signaling in the GLI2+ stromal cells of the Gli2creERT2-tdt:R26RYFP/SmoM2 murine lung.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Whole lung was dissected from the adult mouse and tracheally perfused with a digestion cocktail of Collagenase Type I (225 U/ml, Thermo Fisher), Dispase (15 U/ml, Thermo Fisher) and Dnase (50 U/ml, Sigma) and removed from the chest. The lung was further diced with razor blades and the mixture was incubated for 45 mins at 37 ºC and vortexed intermittently. The mixture was then washed with FACS buffer (2% FBS in DMEM-F12). The mixture was passed through a 70 µm cell strainer and resuspended in RBC lysis buffer, before passing through a 40 µm cell strainer. Cells suspensions were incubated with the appropriate antibodies in FACS buffer for 30 min at 4 ºC and washed with FACS buffer. DAPI (0.2 µg/ml) was used to exclude dead cells. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. YFP+ cells were sorted by endogenous YFP fluorescence. Cells were sorted into FACS buffer. FACS analysis was performed by FACSDiva (BD Biosciences). The RNA was extracted by PicoPure RNA Isolation Kit (Applied Biosystems) from the sorted cells, and the amount and quality of extracted RNA was measured by RNA 6000 Pico Kit (Agilent). The Bulk RNA-seq was done in GENEWIZ.
RNA sequencing library preparation used the NEBNext Ultra RNA Library Prep Kit for Illumina by following manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, mRNA were first enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends,and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA).
The sequencing libraries were multiplexed and clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
FASTQC for quality control, Cuadapt and Sickle for triming adaptors
reads were aligned and mapped using HISAT and Stringtie
DESeq was use to call differential gene expression, in Rstudio
Genome_build: UCSC mus musculus mm10
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| Submission date |
Aug 11, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Peng Tien |
| E-mail(s) |
tien.peng@ucsf.edu
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| Phone |
4155144180
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| Organization name |
University of California San Francisco
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| Department |
Department of Pulmonary Medicine
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| Lab |
Dr. Tien Peng
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| Street address |
513 Parnassus Ave, Health Sciences East 1350
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE102589 |
Hedgehog gradient in the stroma maintains niche diversity and organ function [RNA-seq] |
| GSE102592 |
Hedgehog gradient in the stroma maintains niche diversity and organ function |
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| Relations |
| BioSample |
SAMN07501473 |
| SRA |
SRX3090434 |
| 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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