GSM3209143: Postnatal day 10 Lipochondrocyte 2; Mus musculus; RNA-Seq
RNA from freshly isolated ear cartilage lipochondrocytes or adipocytes was harvested using a modified TRIzol reagent protocol. For stages P8, P10, and P13, ear cartilage from Wnt1-Cre2;TdTomato mice was dissected and digested in 1% Collagenase II in DMEM to generate a single cell suspension. Cells were then collected via FACS using a TdTomato High gate, followed by RNA extraction. For stages P21 and P33, ear cartilage was dissected carefully avoiding adipocytes and digested as described to generate a single cell suspension. Cells were centrifuged at 400g for 5 minutes and the buoyant fraction was collected for RNA extraction. For adipocyte controls, inguinal fat was minced, digested, and centrifuged as described. The buoyant fraction was collected for RNA extraction. For RNA extraction, all tissues were homogenized in TRIzol reagent using a tissue homogenizer after isolation. After homogenization, any lipid layers were avoided by collecting the other fraction of the homogenate.Chloroform was added to the homogenates, followed by vortexing and centrifugation according to protocol. The aqueous phase was collected and ran through a QIAGEN RNeasy micro kit, and the manufacturer's instructions were followed. 1 ug of total RNA was used for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols.