Transcriptome profiling of purified mouse platelets

Purpose: The aim of this study is to determine the relative expression levels of mRNA transcripts in wild type platelets Methods: Total RNA was extracted and purified from purified platelets from BALB/c male mice (3 independent samples). Platelet purification was performed as described in Josefsson EC et al, Journal of Experimental Medicine (2011) 208:2017-31. Total RNA (100 ng) was used to generate sequencing libraries for whole transcriptome analysis following Illumina's TruSeq RNA v2 sample preparation protocol. Completed libraries were sequenced on HiSeq 2000 with TruSeq SBS Kit v3- HS reagents (Illumina) as 100 bp paired-end reads at the Australian Genome Research Facility (AGRF), Melbourne. Reads were aligned to the mouse reference genome mm10 and counts for known genes were obtained using the Rsubread package (version 1.18.0) (Liao et al. 2013; Liao et al. 2014). Total RNA was extracted and purified from purified platelets from BALB/c male mice (3 independent samples per population).研究目的：本研究旨在测定野生型血小板中mRNA转录本的相对表达水平。实验方法：按照Josefsson EC等人发表于《实验医学杂志（Journal of Experimental Medicine）》(2011) 208:2017-31的方案对BALB/c雄性小鼠的血小板进行纯化，随后从纯化后的血小板中提取并纯化总RNA，共设置3份独立样本。取100 ng总RNA，遵循Illumina TruSeq RNA v2样本制备规程构建全转录组测序文库。构建完成的文库在墨尔本的澳大利亚基因组研究中心（Australian Genome Research Facility, AGRF）使用HiSeq 2000测序仪，搭配TruSeq SBS Kit v3-HS试剂，以100 bp双端读长模式完成测序。将测序读段比对至小鼠参考基因组mm10，并通过Rsubread软件包（Rsubread package，版本1.18.0）获取已知基因的计数数据（Liao等，2013；Liao等，2014）。此外，从各群体的BALB/c雄性小鼠纯化血小板中提取并纯化总RNA，每个群体设置3份独立样本。