Spriselect size selection beads

Tandem affinity purifications of FTP-tagged strains were performed as described above with minor modifications. Reagents were prepared under RNase-free conditions, in the presence of RNase inhibitor (Invitrogen, 10777019). Two-third of final flag elute was taken for DNase treatment using NEB DNase 1 (M0303) and RNA was purified using RNA Clean and Concentrator Kit (Zymo Research, R1015/R1017). The final RNA concentration was determined using Qubit RNA high Sensitivity Assay Kit and 100 ng of RIP-purified RNA was used for cDNA library preparation using NEBNext Ultra II directional RNA library kit (NEB, E7760) following library preparation protocol. The final cDNA libraries were amplified using NEBNext Multiplex Oligos for Illumina (NEB, E7335) and purified using SPRIselect size selection beads (Beckman Coulter, B23317). Individual library quality was assessed on TapeStation (Agilent 4200) using a DNA D1000 High sensitivity tape. Indexed libraries were pooled and sequenced on NovaSeq sequencer with 150 bp paired-end reads. Similarly, 200 ng of RIP-purified RNA was used for library preparation using Oxford Nanopore direct RNA sequencing library kit (SQK-RNA002; Version: DRS_9080_v2_revM_14Ag2019) protocol. The prepared libraries were run on individual flow cells following manufacturer guidelines.

Total RNA was isolated, DNase treated and purified from respective strains as mentioned above. The RNA quality was assessed using Bioanalyzer (Agilent 2100) and only those of high quality was proceeded to library preparation. Briefly, 100 ng was taken from individual samples, and an ERCC-RNA spikeIn (Life technologies, 4456740) amount equivalent to that for 1 μg of RNA input (equivalent to that of poly(A) + sample) was added. The RNA samples were directly proceeded for cDNA library preparation using NEBNext Ultra II directional RNA library kit (NEB, E7760) following the library preparation protocol. The final cDNA libraries were amplified (cycle number of 6) using NEBNext Multiplex Oligos for Illumina (NEB, E7335) and purified using SPRIselect size selection beads (Beckman Coulter, B23317). Individual library quality was assessed on TapeStation (Agilent 4200) using a DNA D1000 High sensitivity tape. Indexed libraries were pooled and subjected for deep sequencing with ~80 M reads per sample on a NovaSeq. sequencer with 150 bp paired-end reads.

Total RNA was isolated, DNase treated and purified from respective strains as mentioned above. The RNA quality was assessed using Bioanalyzer (Agilent 2100) and only those of high quality was proceeded to library preparation. Briefly, 1 μg was taken from individual samples, and ERCC-RNA spikeIn (Life technologies, 4456740) was added according to manufacturer’s instructions. The RNA samples were then subjected to OligodT purification of poly(A) RNA (NEB, E7490) following the manufacturer’s instructions and the recovered poly(A) selected RNA was used for cDNA library preparation using NEBNext Ultra II directional RNA library kit (NEB, E7760) following library preparation protocol. The final cDNA libraries were amplified (cycle number of 8) using NEBNext Multiplex Oligos for Illumina (NEB, E7335) and purified using SPRIselect size selection beads (Beckman Coulter, B23317). Individual library quality was assessed on TapeStation (Agilent 4200) using a DNA D1000 High sensitivity tape. Indexed libraries were pooled and sequenced with ~20 M reads per sample on a NovaSeq. sequencer with 150 bp paired-end reads.