GSM2195200: NIPP1_KO_testis_mouse2; Mus musculus; RNA-Seq
Adult testes were harvested 2 weeks after treatment with tamoxifen. RNA extracted from 40 mg of testis; GenEluteTM Mammalian Total RNA Miniprep kit (SIGMA). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 5 µg of total RNA was used as input. Using the Illumina TruSeq® Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (protocol version Rev E. “October 2013”) rRNA is depleted from the total RNA samples using Ribo-Zero ribosomal RNA reduction chemistry. Subsequently, RNA was purified and fragmented and converted into first strand cDNA in a reverse transcription reaction using random primers. Next, double-stranded cDNA was generated in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a  single ‘A’ base to the 3’ ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichmend PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence-libraries of each sample were equimolarly pooled and sequenced on ½ run of a NextSeq500 flow-cell at 2x75 bp.