title: GSM3463241: DMS-MaPseq on in vivo Influenza A vmRNA probe pairing regions (Replicate #1); Influenza A virus; OTHER
library_construction_protocol: 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 using the TGIRT-III reverse transcriptase 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. Primers annealed directly before the respective probe pairing position and contained an overhang corresponding to the standard Illumina adapter . cDNA was PCR-amplified and the resulting amplicons were pooled equimolarly to 1 ng/µl. Indexed adapter sequences were further added by a second PCR enrichment step.