Ercc rna spikein

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.

Total RNA-Seq analysis was performed from two biological replicates of YAM2478 (WT), YAM2479 (xrn1Δ), YAM2795 (dcr1Δ), and YAM2796 (dcr1Δ xrn1Δ) cells. For each sample, 1 μg of total RNA was mixed with 2 μl of 1:100 diluted ERCC RNA spike-in (Life Technologies), then ribosomal (r)RNAs were depleted using the RiboMinus Eukaryote v2 Kit (Life Technologies). Total RNA-Seq libraries were constructed from 50 ng of rRNA-depleted RNA using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina). Paired-end sequencing (2 × 50 nt) was performed on a HiSeq 2500 system (Illumina).
The N. castellii reference genome was retrieved from version 7 of the Yeast Gene Order Browser (Byrne & Wolfe, 2005 (link)); snoRNAs were annotated using the S. cerevisiae snoRNAs as queries for blastn alignments (E value cutoff e⁻⁸). Reads were mapped using version 2.0.9 of TopHat (Kim et al, 2013 (link)), with a tolerance of three mismatches and a maximum size for introns of 2 Kb. All bioinformatics analyses used uniquely mapped reads. Tag densities were normalized on the ERCC RNA spike-in signal.