GSM3463240: DMS-MaPseq on in vivo spliced segment 7/8 Influenza A vmRNAs (Replicate #2); Influenza A virus; OTHER
Cells were collected by centrifugation and total RNA extracted with TRIzol Reagent (ThermoFisher Scientific, #15596018). After phase separation, the aqueous phase was added to 1 ml of 100% ethanol and the RNA purified on RNA Clean & Concentrator-5 columns (Zymo Research, #R1016). RT reactions were performed in 20 µl total reaction volume. An anchored oligo(dT) primer with overhang for subsequent PCR was used at a final concentration of 50 µM. The reaction was allowed to proceed at 57 °C for 2 h. Afterwards, RNA was degraded by adding 1 µl 5 M NaOH and heating to 95 °C for 3 min and subsequently purified on RNA Clean & Concentrator-5 columns. Short splice isoforms were then specifically amplified via PCR and gel purified. The short splice isoforms were mixed equimolarly and fragmented by sonication to yield fragments in the range of 100-300 bp. Sonicated DNA was gel purified to remove residual full-length products and subjected to library preparation using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs, #E6240L) according to manufacturer's instructions, but omitting size selection.