We then sorted the candidate hairpin sequences by score, breaking high scoring ties by the total number of lincRNA isoforms that are covered by the hairpin. We then aligned each hairpin sequence against both the genome and the RefSeq-defined transcriptome (NCBI Release 39), and filtered any hairpin with fewer than three mismatches to any other gene or position in the genome. Candidate sequences were chosen for shRNA production by first picking the highest scoring candidate and then proceeding to successively lower scores. As each hairpin was selected, all other hairpins overlapping this hairpin were removed. We repeated this process until we identified 5 hairpins that covered each lincRNA.
