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Sample GSM2883477

Query DataSets for GSM2883477

Status

Public on May 29, 2018

Title

R1_RNA_+iFGFR1

Sample type

SRA

Source name

Mammary epithelial cells

Organism

Mus musculus

Characteristics

strain: FVB/N
tissue: Mammary gland
age: 4-week old
genotype: MMTV-Wnt1 / MMTV-iFgfr1

Treatment protocol

4-week old MMTV-Wnt1 / MMTV-iFgfr1 female mice were treated i.p. with vehicle (-iFGFR1) or AP20187 (Clontech, 635069) for 6 hours to activate iFGFR1 signaling (+iFGFR1).

Extracted molecule

total RNA

Extraction protocol

The 3rd, 4th and 5th mammary glands were isolated from mice with and without iFGFR1 activation and immediately transferred to DMEM/F-12 (Thermo Scientific, 11330032) media with 100 μg/ml CHX (Sigma-Alrich, C4859) at room temperature. The glands were then chopped into small pieces and subsequently digested in 1 mg/ml Collagenase A (Sigma-Alrich, 11088793001) in DMEM/F-12 with 100 μg/ml CHX for 2 hrs at 37 °C and 125 rpm rotation. Pieces of glands were resuspended up and down every 30 min to break up aggregates, and 0.5 μg/ml DNase I (StemCell Technologies, 07900) was added 30 min prior to MEC harvest. Cell suspensions were pelleted at 1500 rpm for 5 min. MECs were then purified with serial centrifugation by washing twice in PBS containing 5% FBS (Thermo Scientific, 10437010) and 100 μg/ml CHX with at 1500 rpm for 7 s. The cell pellet was subsequently processed for purification of ribosome protected fragments (RPFs) and RNA. RNA was harvested using TRIzol Reagent in accordance with manufacturer’s protocol (Thermo Scientific, 15596018). RPFs were isolated using a published protocol (Ingolia et al., 2012; PMID:22836135) with a few modifications. Instead of sucrose gradient centifugation, recovery of RPFs following nuclease treatment was performed using Illustra MicroSpin S-400 HR columns (GE Healthcare, 27514001) and the RNA Clean & ConcentratorTM-25 kit (Zymo Research, R1017) as described in the protocol of TrueSeq® Ribo Profile kit (Illumina, RPHMR12126).
RNA-Seq libraries were prepared for sequencing using TruSeq® Stranded mRNA Library Prep Kit Set A (Illumina, RS1222101). Ribo-Seq libraries were prepared as described in "extract protocol" section.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Base-calling was performed using Real Time Analysis (RTA) v.1.18.64. File conversion and demultiplexing were conducted using bcl2fastq-1.8.3.
The adapter sequences were removed from the Ribo-Seq and RNA-Seq reads using Cutadapt. To remove rRNA contamination, both Ribo-Seq and RNA-Seq reads were first mapped to the mouse rRNA library. rRNA contaminating reads were then filtered out, and rRNA-free reads were extracted for further analysis.
For RNA-Seq, the rRNA-free reads were mapped to the mouse genome mm10 using STAR. For Ribo-Seq, rRNA-free reads were first mapped to the mouse genome mm10 using Bowtie2. The unmapped reads were extracted and then mapped to mm10 using STAR. The Bowtie2 and STAR mapped reads were then merged. HTSeq was used to count the reads mapped to the known genes for both RNA-Seq and Ribo-Seq.
Genome_build: mm10
Supplementary_files_format_and_content: csv file includes read counts for each sample

Submission date

Dec 11, 2017

Last update date

May 15, 2019

Contact name

Jeffrey M Rosen

Organization name

Baylor College of Medicine

Department

Molecular and Cellular Biology

Street address

1 Baylor Plaza