GSM3385692: BAL_Control [Chris_G_A3_19_WB11_14_S208_L006]; Mus musculus; RNA-Seq
BAL Cell pellets were obtained from pooled lavage fluid, washed once in PBS and lysed in 350 μl RLT buffer. Lysates were passed through QIAshredder columns (QIAGEN). RNA was extracted using the RNeasy mini kit as per manufacturer's instructions, including on-column DNase I digestion (both QIAGEN). Blood was collected in Tempus reagent (Life Technologies) at 1:2 ratio. Total RNA was extracted using the MagMAX™ Blood RNA isolation Kit (Thermo Fisher). Globin RNA was depleted from total RNA (1.5–2 µg) using the Mouse GLOBINclear kit (Ambion). Total/globin-reduced RNA (200 ng) was used to prepare cDNA libraries using the TruSeq Stranded mRNA HT Library Preparation Kit (Illumina). Quality and integrity of the tagged libraries were initially assessed with the HT DNA HiSens Reagent kit (Perkin Elmer) using a LabChip GX bioanalyser (Caliper Life Sciences/Perkin Elmer). Tagged libraries were then sized and quantitated in duplicate (Agilent TapeStation system) using D1000 ScreenTape and reagents (Agilent). Libraries were normalised, pooled and then clustered using the HiSeq® 3000/4000 PE Cluster Kit (Illumina). The libraries were imaged and sequenced on an Illumina HiSeq 4000 sequencer using the HiSeq® 3000/4000 SBS kit (Illumina) at a minimum of 25 million paired-end reads (75 bp/100 bp) per sample.
GEO Accession
GSM3385692