GSM3895542: Bin1-WT-M-0177; Mus musculus; RNA-Seq
Qiagen All Prep kit (Cat: 80224, Qiagen) was used for RNA extraction from previously frozen microglia cells. Briefly, cells were lysed with RLT Buffer (with β-mercaptoethanol added), and passed through QIA shredder (Cat: 79654, Qiagen). Flow-through from the QIA shredder was then passed through a DNA Mini column to remove the DNA from the lysate. DNA mini columns were stored at 4°C for DNA purification later; and the passed-through lysates were treated with proteinase K. Following treatment, 100% ethanol was added to the lysates and passed through a RNeasy Mini spin column. In-column DNase treatment was also performed. Subsequently, RNA was eluted with RNase-free water, quantified in Nano-drop and stored at -70°C until samples were submitted for sequencing. 500 ng RNA was used as starting input to Illumina TruSeq mRNA stranded library construction according to the manufacturers recommendation. High quality libraries were then pooled and sequenced on an Illumina HiSeq 2500 cDNA from each sample was generated from 5ng of total RNA using SMARTer Ultra Low Input RNA kit v4 (Clontech).  The cDNA was amplified by 9 PCR cycles, followed by QC analysis on BioAnalyzer 2100 (Agilent).  Sequence libraries were produced from 150pg of cDNA using Nextera XT DNA library kit (Illumina), cleaned up with AMPure XP beads, and QC checked with Caliper LabChip GX.  Paired-end sequencing data were generated on an Illumina HiSeq 2500, at a depth of 20 million reads per sample, with read length 50b X 2.