GSM5597849: P7_L1L2AKO_adipocyte_rep 2; Mus musculus; RNA-Seq
Adipocytes were isolated from four pooled male P7 L1L2-FF or L1L2-AKO mice. Inguinal scWAT were minced carefully with scissors for 3-5 min and digested with 4 mg/ml Collagenase Type I (in 4 mL PBS containing 1% BSA) at 37°C with agitation at 170 rpm for 12-15 min. The digested cell suspension was quenched in 4 mL PBS containing 1% BSA, followed by free floating for 5 min. After removing infranatant with a syringe and needle, floated cells were filtered using a 300 μm strainer and washed with 3 mL PBS containing 1% BSA. Floated adipocytes were collected by centrifugation at 30 × g for 5 min and lysed with Trizol reagent for total RNA extraction. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library.
GEO Accession
GSM5597849