title: GSM5553738: Bulk sedentary standard (chow) diet rep2 [SC4.scWAT]; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted at the Goodyear lab using an RNA extraction kit (Direct-zol™ RNA MiniPrep, Zymo Research). ​​10ng of total RNA quantified and quality assessed by Advanced Analytical Fragment Analyzer was used for library preparation on Tecan Evo150. 3′ DGE-custom primers 3V6NEXT-bmc#1-12 were added to a final concentration of 1 uM. to generate two subpools of 15 samples each. After addition of the oligonucleotides, Maxima H Minus RT was added per manufacturer's recommendations with Template-Switching oligo 5V6NEXT followed by incubation at 42℃ for 90' and inactivation at 80℃ for 10'. Following the template switching reaction, cDNA from 12 wells containing unique well identifiers were pooled together and cleaned using RNA Ampure beads at 1.0X. cDNA was eluted with 17 ul of water followed by digestion with Exonuclease I at 37℃ for 30 minutes, and inactivated at 80℃ for 20 minutes. Second strand synthesis and PCR amplification was done by adding the Advantage 2 Polymerase Mix (Clontech) and the SINGV6 primer directly to the exonuclease reaction. Eight cycles of PCR were performed followed by clean up using regular SPRI beads at 0.6X, and eluted with 20ul of elution buffer (Qiagen). Successful amplification of cDNA was confirmed using the Fragment Analyzer. Illumina libraries were then produced using standard Nextera tagmentation substituting P5NEXTPT5-bmc primer in place of the normal N500 primer. Final libraries were cleaned using SPRI beads at 0.7X and quantified using the Fragment Analyzer and qPCR before being loaded for sequencing using the Hiseq 2000 (Illumina, Inc) in 50bp single-end mode at the BioMicro Center at MIT. 3′ Digital Gene Expression (DGE) RNA-seq