GSM7816350: iWAT WT control for H2KO; Mus musculus; RNA-Seq
Subcutaneoues white fat pad were removed, flash frozen in liquid nitrogen, and RNA was harvested using Tri-reagent After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes). 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. After agarose gel electrophoresis, 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. At last, the library could be sequenced using Illumina HiSeqTM 2000 or other sequencer when necessary