title: RNA-Seq of Homo sapiens: bulk bone
design: All the prepared libraries were analyzed in a single batch. TruSeq RNA Library prep kit V2 (Illumina, USA) was used to capture poly(A) RNA from 1000ng total RNA. Subsequently cDNA was prepared to which single indexed adapters were ligated. Material was then cloned for 13 cycles with PCR. Paired-end sequencing of 2x50 bp was performed using the Illumina HiSeq2000 (Illumina, USA) platform to obtain at least 6.000.000 reads per library. They were aligned to reference genotype (GRCh37.p13, gencode release 19) using STAR 2-pass methodology. Picard tools were used to add readgroups to bam files, reorder contigs according to the reference file and remove PCR duplicates. In the next step, GATK was used to split cigar reads, realign reads around indels, and recalibrate quality scores of the resulting reads. Picard was again used to extract QC metrics .