2200 tapestation with high sensitivity d1000 reagent kits

RNA sequencing was performed using SMART-3Seq, a 3' tagging strategy specifically designed for degraded RNA directly from FFPE LCM specimen^{27 (link)}. LCM dissected SMART-3Seq libraries were prepared using the standard protocol for FFPE tissue on Arcturus HS LCM Cap and the individual library SPRI purification option. All FFPE LCM dissected libraries were amplified using 19 PCR cycles during indexing to minimize over-amplification of high abundance mRNAs in each library. Libraries were individually analyzed for size distribution on an Agilent 2200 TapeStation with High Sensitivity D1000 reagent kits to verify average library size of 190 bp and stored at −20 °C until sequencing. When all libraries were ready for sequencing, 1 µL of each library was then used to create two library pools used for sequencing and quantified by Qubit 2.0 Fluorometer HS DNA assay. Library pools were sequenced with a 1% PhiX spike-in control library and sequenced on an Illumina HiSeq 4000, a run type of single read 75 (SR75), and dual index sequencing.

Smart-3SEQ sequencing libraries from FFPE cells were prepared and pooled according to version 1.9 of the Smart-3SEQ protocol [11 (link)]. Briefly, the pre-SPRI pooling option was used for a single batch for all LCM samples, and 22 PCR cycles were used for library amplification. Libraries were profiled for size distribution on an Agilent 2200 TapeStation with High Sensitivity D1000 reagent kits and quantified by qPCR with a dual-labeled probe [13 (link)], and libraries were mixed to equimolarity according to the qPCR measurements. Libraries were sequenced with Illumina NextSeq 500 instrument with a High Output v2 reagent kit (Illumina FC-404-2005). The 76-base directional, single-end sequencing reads were obtained and uniquely mapped to hg38 using STAR with the settings provided in the previous protocol [11 (link)].