RNA-seq from FFPE Tissue

RNA was purified from FFPE tissue using the MagMAX FFPE DNA/RNA Ultra kit, reverse-transcribed using the NEBNext RNA First Strand Synthesis Module (cat# E7525S, NEB) and NEBNext Ultra II non-directional RNA Second Strand Synthesis Module (cat# E6111S, NEB) for cDNA Synthesis. RNA-seq libraries were prepared from the cDNA using SureSelect XT HS and Low Input Library Preparation Kit for ILM (Pre PCR) (cat# G9704, Agilent, Santa Clara, CA, USA) following the manufacturer’s instruction. The libraries were sequenced on an Illumina NovaSeq-6000 Sequencing System to generate 150 × 150 paired-end reads. Using FFPE sample for RNA-Seq is a more difficult task than using fresh or snap-frozen tissue. However, FFPE is still the most accessible tumor samples so studies have been lunched to test the use of RNA from FFPE sample for RNA-seq analysis and established reliable protocols [16 (link), 17 (link)]. The protocols have since been adopted to routine use in oncogenomics studies and commercial kits have been developed by several suppliers, including the two New England Biolabs kits (NEBNext RNA First Strand Synthesis Module and NEBNext Ultra II non-directional RNA Second Strand Synthesis Module) we used above.