Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (#74704, Qiagen). RNA libraries for sequencing were prepared using poly-A selection and the Illumina RNA strand-specific TruSeq Stranded mRNA Sample prep kit with 96 dual indexes (Illumina, CA, USA) according to the manufacturer’s instructions with the following changes. The protocols were automated using an Agilent NGS workstation (Agilent, CA, USA) using purification steps as described56 (link),57 (link).
Clustering was done by ‘cBot’ and samples were sequenced on HiSeq. 2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2 × 125/2 × 150 setup using ‘HiSeq SBS Kit v4’ chemistry. The Bcl to FastQ conversion was performed using bcl2fastq-v2.17.1.14 from the CASAVA software suite. The quality scale used was Sanger/phred33/Illumina 1.8 + .
Clustering was done by ‘cBot’ and samples were sequenced on HiSeq. 2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2 × 125/2 × 150 setup using ‘HiSeq SBS Kit v4’ chemistry. The Bcl to FastQ conversion was performed using bcl2fastq-v2.17.1.14 from the CASAVA software suite. The quality scale used was Sanger/phred33/Illumina 1.8 + .
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