GSM4804561: Setd7 WT Kidney - Diabetic 374; Mus musculus; RNA-Seq
Drop-Seq. Kidney cortex was dissected and homogenized by pressing though a 100uM sieve and glomerular fraction collected using 70uM sieve. Glomerular fraction was dissociated using cold active protease (subtilisin A, Sigma). Single cell RNA was captured using the drop-seq method (Macosko 2015) Drop-Seq. Sequencing libraries were created from barcoded cDNA (Drop-seq) using the illumina Nextera XT library prep kit (Illumina) as adapted in the Drop-Seq protcol (Macosko 2015) RNA-Seq. Kidneys were homogenized TRIzol solution. RNA was extracted using the Directzol RNA mini-prep kit according to the manufacturer's protocol, with DNase I treatment in the column to remove genomic DNA contamination (Zymo Research, Irvine, CA, USA). RNA was eluted in 30 μl TE buffer, and the quantity and quality of RNA examined on a MultiNA bioanalyzer (Shimadzu, Tokyo, Japan). RNA-Seq. A NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA) was used to enrich mRNA from 1 μg of total RNA. The NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® was then used to generate barcoded libraries according to the manufacturer's protocol, and these libraries were then quantified on the MultiNA bioanalyzer and pooled to equimolar ratios for sequencing.