GSM8416984: ST_PRV_UL13null_rep2; Sus scrofa; Suid alphaherpesvirus 1; OTHER
RNA isolations were performed using the RNeasy minikit (Qiagen) according to the manufacturer's procedure. Purified RNA was treated with RNase-free DNase I (New England Biolabs) at 37°C for 10 min to remove contaminating DNA. To stop DNase I activity, EDTA (Invitrogen) was added at a final concentration of 5 mM and samples were incubated at 75°C for 10 min. CROWN-seq is performed as previously described with some modifications. ~30 μg total RNA input was used for the CROWN-seq libraries. The RNA input was converted by sodium nitrite as described in the GLORI protocol1. After conversion, the m7G capped 5' end RNA fragments were isolated based on a modified ReCappable-seq workflow2. In brief, the converted m7G capped RNAs were firstly recapped by 3´-Desthiobiotin-GTP. Then the recapped RNA fragments were enriched on streptavidin beads. Different from the original ReCappable-seq protocol, we performed on-beads 3' adapter ligation, where a barcoded 3' DNA adapter (/rApp/ANNNNNNNNXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACAAAAAAAAAAAAAAACCCCCCCCCCAAAAAAAAAAAAAAA/3AmMO/, X=barcode) was ligated to the enriched RNA fragments. We used four different barcodes to label different samples. After adapter ligation, 2-4 samples in different barcodes were pooled. This pooling strategy can increase the yield of library. The pooled RNA fragments were then treated by 5´ Deadenylase (NEB #M0331S) and RecJf (NEB #M0264S), which can remove the exceeded 3' adapter. After a second round streptavidin enrichment, the RNA fragments were eluted into water. To ligate 5' adapter, we used RppH (NEB #M0356S) to release the 3´-Desthiobiotin caps, which results in monophosphate 5' ends readily for ligation. We then ligated a 5' adapter (rCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrArUrNrN) which contain unique molecular identifier (UMI) to the RNA fragments. After adapter ligation, reverse transcription primer (GACGTGTGCTCTTCCGATCT) was annealed to the 3' adapter (75°C 5 min, 37 °C 15 min, then 25 °C 15 min). Reverse transcription was performed with SuperScript III (Thermo Fisher #18080093). After reverse transcription, cDNA was amplified with Phusion PCR master mix (NEB #M0531S) and NEBNext multiplex oligos (NEB #E7780S). Amplified libraries were size selected by SPRIselect beads (Beckman #B23317). Size selected libraries were then sequenced by NovaSeqX in PE100 mode.