title: GSM4801772: H7-C2; Homo sapiens; RNA-Seq
library_construction_protocol: The human islets were lysed with 350 uL of QIAzol Lysis Reagent (Qiagen) and stored at -80˚C before purifying RNA using Mini RNeasy purification kits per manufacturer's instructions. Total RNA submitted to the University of Wisconsin-Madison Biotechnology Center was verified for purity and integrity via the NanoDrop2000 Spectrophotometer and Agilent 2100 BioAnalyzer, respectively. Samples that met the Illumina sample input guidelines were prepared according the TruSeq® Stranded Total RNA Sample Preparation Guide (Rev. E) using the Illumina® TruSeq® Stranded Total RNA Sample Prep Gold kit (Illumina Inc., San Diego, California, USA) as directed. For each library preparation, 1ug of total RNA was ribosomally reduced and purified by paramagnetic beads (Agencourt RNA Clean XP beads, Beckman Coulter, Indianapolis IN, USA). Subsequently, each rRNA depleted sample was fragmented using divalent cations under elevated temperature. The fragmented RNA was synthesized into double-stranded cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, California, USA) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA Polymerase I and RNase H for removal of mRNA. Double-stranded cDNA was purified by paramagnetic beads (Agencourt AMPure XP beads, Beckman Coulter). The cDNA products were incubated with Klenow DNA Polymerase to add an 'A' base (Adenine) to the 3' end of the blunt DNA fragments. DNA fragments were ligated to Illumina adapters, which have a single 'T' base (Thymine) overhang at their 3'end. The adapter-ligated DNA products were purified by paramagnetic beads. Adapter ligated DNA was amplified in a Linker Mediated PCR reaction (LM-PCR) for 10 cycles using PhusionTM DNA Polymerase and Illumina's PE genomic DNA primer set and then purified by paramagnetic beads. Quality and quantity of the finished libraries were assessed using the AATI Fragment Analyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) and BioPlex Synergy 2 Plate Reader (BioTek® Instruments, Winooski, VT, USA), respectively.