GSM1961239: Lgr6- rep2; Mus musculus; RNA-Seq
Mammary glands from PDL mice were dissected, mashed gently with a MACS dissociator (Miltenyi Biotec) and incubated in HBSS with 300U ml-1 collagenase (Sigma) plus 300 μg hyaluronidase (300U ml-1activity) (Sigma) and digested for 30m at 37°C. Red blood cells were lysed and samples digested with Trypsin for 3 min at 37°C before being filtered with a 70 by 40 μm-mesh. LIN- cells where then FACS sorted for LGR6+ cells. RNA concentrations were determined with a Qubit fluorometer (ThermoFisher) as 7-25 pg/ul before analysis of RNA quality on a Bioanalyzer (Agilent) (RIN 5-8). cDNA (50-75ng/μl) was generated from 5μl RNA (NuGEN/Next-Gen), sheared (Covaris S-series), and cDNA fragments were cleaned-up (Zymo Research) before 10ng were used to generate an amplified cDNA library (NuGEN Ovation) over 18 PCR cycles. cDNA was sequenced on a HiSeq 4000 system (illumina) in paired-end 50 mode
GEO Accession
GSM1961239