GSM2342532: Normal Sample 2; Mus musculus; RNA-Seq
For RNA-seq, we used 13 kidney tissue samples from wild-type mice, 7 from the diabetic Ins2Akita mice, 8 from early P78 treatment of diabetic Ins2Akita mice, and 7 from late P78 treatment the diabetic Ins2Akita 7 mice. Total RNA was extracted using mirVana kit (Life Technologies) with some modifications. Briefly, a bead mill homogenizer (Bullet Blender, Next Advance) was used to homogenize the tissue using a safe-lock microcentrifuge tube (Eppendorf) and a mass of stainless steel beads (Next Advance, cat# SSB14B) equal to the mass of the tissue. Immediately after two volumes of lysis buffer were added to the tube, samples were mixed in the Bullet Blender for 1 min at a speed of six. Samples were visually inspected to confirm desired homogenization and then incubated at 37 °C for 5 min. The lysis buffer was added up to 0.6 ml, and samples were mixed in the Bullet Blender for 1 min. Optical density values of extracted RNA were measured using NanoDrop to confirm an A260:A280 ratio above 1.9. RNA integration number (RIN) was measured using BioAnalyzer (Agilent) RNA 6000 Nano Kit to confirm RIN above 7. cDNA libraries were prepared using SureSelect Strand Specific RNA Library Preparation Kit (Agilent) as per the manufacturer's instructions. Briefly, polyA RNA was purified from 1000 ng of total RNA using oligo (dT) beads. Extracted RNA was subjected to fragmentation, reverse transcription, end repair, 3'-end adenylation, adaptor ligation and subsequent PCR amplification and SPRI bead purification (Beckman Coulter). The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent) and Kapa Library Quantification Kit (Kapa Biosystems). 12 libraries were pooled and diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The denatured libraries were diluted to 10 pM by pre-chilled hybridization buffer and loaded onto TruSeq SR v3 flow cells on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe (TruSeq SBS Kit v3) according to the manufacturer's instructions (Illumina).
GEO Accession
GSM2342532