NextSeq 500 paired end sequencing; RNA-seq of SOX2 positive pituitary cells with and without activation of beta-catenin
RNA-seq of SOX2 positive pituitary cells with and without activation of beta-catenin
Sox2CreERT2/+; Ctnnb1lox(ex3)/+; ROSA26flox-stop-YFP/+ and Sox2CreERT2/+; ROSA26flox-stop-YFP/+ mice were induced with a single intra-peritoneal injection of tamoxifen (Sigma) at a dose of 0.15 mg per gram of body weight at 4 weeks of age. 1 month post-induction mice were culled and Anterior pituitaries were dissociated mechanically into a single cell suspensions following incubation in an enzyme mix (0.5% w/v Collagenase type 2 (Lorne Laboratories Ltd), 0.1X Trypsin (Gibco) and 50 μg/ml DNaseI (Worthington) with 2.5 μg/ml Fungizone (Gibco) in Hank's Balanced Salt Solution (HBSS (Gibco))) for 4 hours at 37°C. Cells were then washed in HBSS and suspended in PSB supplemented with 1% Fetal Calf Serum (PAA), 25mM HEPES and 1 ng/μl of propidium iodide (PI (BD Pharmigen)). Cells were flow sorted for YFP and PI fluorescence using a MoFlo XPD (Beckman Coulter, Fullerton, CA, USA) Flow Cytometer. Fluorescence was detected using a 530/540 filter, where PI positive cells were excluded and the YFP positive and negative fractions collected directly into RLT buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Sigma). The SMARTer v4 low input assay kit (Clontech) was used to create amplified cDNA using the strand-switching protocol. Flow sorted cells from Sox2CreERT2/+; Ctnnb1lox(ex3)/+; ROSA26flox-stop-YFP/+ and Sox2CreERT2/+; ROSA26flox-stop-YFP/+ mice were processed for total RNA extraction using the RNeasy Micro kit (Qiagen). cDNA library preparation was performed using 200pg of amplified cDNA with the Nextera XT preparation kit (12 cycles of PCR).