GSM2883474: R1_RNA_-iFGFR1; Mus musculus; RNA-Seq
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.
GEO Accession
GSM2883474