Nebnext ultra 2 directional rna library kit

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.

For each strain, RNA extraction of two biological replicates was performed. The cells were grown till the mid-exponential phase and then 50 ml cells were harvested by centrifugation. RNA extraction was done using the TRIzol (Invitrogen)-chloroform method as previously described [44 (link),95 (link)]. DNase treatment was done to remove any DNA contamination after which enrichment for mRNA was done using the MicrobExpress Kit (Invitrogen) following the manufacturer's protocol. Single-end, strand-specific libraries for RNA sequencing were prepared using NEBNext Ultra II Directional RNA library kit (New England Biolabs). The quantity of the mRNA was assessed using Multiscan GO (Thermo Scientific) Nanodrop and the quality, as well as integrity, was checked using BioAnalyzer. The sequencing was carried out on HiSeq 2500 Rapid Run Mode using a 1 × 50 bp format.

Yeast strains transformed with an empty URA vector, pRS426, were grown at 30°C in SD-URA + 2% glucose media to an OD₆₀₀ of 0.5. Cultures were then treated with 4-thiouracil at a final concentration of 0.32 mg/ml for 10 min at 30°C. Cultures were fast cooled in a dry ice/ethanol bath before pellets were harvested by centrifugation and stored at −80°C. RNA was extracted using a hot phenol protocol. RNAs were sonicated to an average size of 1 kb in a Covaris sonicator. 4-Thiouracil-containing RNAs were biotin-labeled using EZ-link biotin (Sigma) at a final concentration of 0.2 mg/ml for 2 h at room temperature. Treated RNAs were extracted twice with chloroform and ethanol precipitated. Biotinylated RNAs were enriched using the μMACS Streptavidin kit (Miltenyi) as per manufacturer's instructions. Enriched RNAs were ribodepleted using a Ribominus yeast/bacteria ribodepletion kit (Thermofisher) and size selected using an RNA Clean and Concentrator-5 kit (Zymo Research). Libraries were prepared using the NEBNext Ultra II Directional RNA Library kit (NEB) as per manufacturer's directions. Sequencing was performed on an Illumina NovaSeq (Novogene).

Total RNA was extracted from 2 mL of E. coli DY330 culture exponentially growing in LB to OD₆₀₀ = 0.6 using ExtractRNA reagent (Evrogen). Samples were treated with DNase I (Thermo Fisher Scientific) and purified by RNAClean XP beads (Beckman Coulter). Sequencing libraries were prepared without rRNA depletion using NEBNext Ultra II Directional RNA Library kit (NEB) with the following modifications: 10 min of fragmentation and ten PCR cycles. The libraries were sequenced on HiSeq 4000 instrument (Illumina, USA) with a 50 bp-long reads protocol. Initial processing of sequencing data (base-calling) was performed with Illumina software HCS v3.3.76 pre-installed in Illumina HiSeq 4000 with standard parameters. Library preparation and sequencing were performed at Skoltech Genomics Core Facility. RNA-Seq was performed in triplicate.
Reads were prepared and mapped to the reference genome as described above for WGS and ChIP-Seq. RSeQC package was used for FPKM and genes expression level calculation^{88 (link)}.

CRISPR-edited and control cell lines (1 × 10⁶ to 2 × 10⁶) were harvested from six-well plates in triplicate in TRI reagent (Sigma Aldrich), and RNA was isolated using Direct-zol miniprep columns (Zymo) according to the manufacturer's specifications. mRNA libraries for Illumina sequencing were prepared using poly(A) mRNA magnetic isolation module and NEBNext Ultra II directional RNA library kits (New England Biolabs) from 250 ng of total RNA. Libraries were sequenced on an Illumina Nextseq 500 instrument to a minimum of 2 × 10⁷ paired-end 75-bp reads. Raw sequencing reads were mapped to the mm10 genome using annotations from the gencode vM10 assembly with STAR version 2.5.0 (Dobin et al. 2013 (link)) using default parameters. Individual transcripts annotated in the gencode vM10 assembly were quantified using featureCounts version 1.5.3 (Liao et al. 2014 (link)) and the option “–t exon” was specified to count mRNA features.