title: GSM7503580: islets of Langerhans-44% fat+fiber-C57BL/6J-F (ORSAM8075) 2; Mus musculus; RNA-Seq
library_construction_protocol: KingFisher Flex purification system (ThermoFisher). Frozen tissues were pulverized using a Bessman Tissue Pulverizer (Spectrum Chemical) and homogenized in TRIzol (ThermoFisher Scientific) using a gentleMACS dissociator (Miltenyi Biotec Inc). After the addition of chloroform to the TRIzol homogenate, the RNA-containing aqueous layer was removed for RNA isolation, according to the manufacturer's protocol, beginning with the RNA bead binding step. RNA was isolated from pancreatic islets using the RNeasy Micro kit (Qiagen), according to manufacturers' protocols, including the optional DNase digest step. RNA concentration and quality were assessed using the Nanodrop 8000 spectrophotometer (Thermo Scientific) and the RNA 6000 Pico or RNA ScreenTape assay (Agilent Technologies). 2 ul of diluted (1:1000) ERCC Spike-in Control Mix 1 (Ambion by Life Technologies) was added to 100ng of each RNA sample prior to library construction.  Libraries were constructed using the KAPA mRNA HyperPrep Kit (Roche Sequencing and Life Science), according to the manufacturer's protocol.  Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification.  The quality and concentration of the libraries were assessed using the D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher), respectively, according to the manufacturers' instructions. Libraries were sequenced on an Illumina NovaSeq 6000 using the S4 Reagent Kit.  Pancreatic islet libraries were sequenced 150 bp paired-end to a targeted read depth of 100 million read pairs.