differential gene expression analysis of Tmem67 knock-out mouse cerebullum
Cerebullum tissue microdissected from Tmem67+/+ wild-type and Tmem67-/- mutant early post-natal (P0) mouse pups. Total RNA was extracted from tissue using standard guanidinium thiocyanate and phenol chloroform extraction (TRIzol, Invitrogen Inc.) RNA samples were treated with a TURBO DNA-free™ Kit (Ambion Inc.) using conditions recommended by the manufacturers, and then cleaned with an RNA Clean & Concentrator™-5 spin column (Zymo Research Corp.) RNA was tested for quality and yield using a NanoDrop 1000 spectrophotometer and an Agilent 2100 Bioanalyzer. RNA was processed further only if samples were uncontaminated with phenol and the RNA Integrity Number (RIN) was >9.0. To minimize bubble PCR artifacts, we used 100 ng of purified total RNA in library preparation, following the “TruSeq” Illumina protocol. In brief, RNA was polyA-selected, chemically fragmented to about 200 nt in size, and cDNA synthesized using random hexamer primers. Each individual library received a unique Illumina barcode. RNA-seq was performed on an Illumina HiSeq 2000 instrument with six libraries multiplexed per flow-cell lane using 100 bp paired-end reads. This resulted in an average of 250 million reads per lane, with an average of 40 million reads per sample.  Raw reads were aligned to the mouse (Mus musculus) full genome (UCSC mm10) using TopHat/Bowtie, a splice-aware aligner. GTF transcript annotation files were downloaded from http://tophat.cbcb.umd.edu/igenomes.html Transcripts were assembled using Cufflinks , followed by estimates of values for isoform-level relative abundance calculated as Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). Differenntial gene expression was analyzed using Cuffdiff, ], with statistically significance expressed as a p-value adjusted for a false discovery rate of 0.01 (q-value) using Benjamini-Hochberg correction for multiple-testing.