T4 rna ligase buffer

Small RNAs (50 ng) were mixed with an 80-mer spike-in RNA oligonucleotide internal standard (DNA and RNA oligonucleotide sequences are detailed in Supplementary Table 2). The mixture was dephosphorylated in a 5 μl reaction containing 0.5 μl of reaction buffer (NEB T4 RNA ligase buffer) and 1 U of shrimp alkaline phosphatase (rSAP, NEB) at 37 °C for 30 min. The reaction was stopped by heat inactivation at 65 °C for 5 min followed by cooling on ice. Linker 1 ligation was performed by adding the following reagents directly to the dephosphorylation product: 1 μl Linker 1 (100 pmol/ul; Supplementary Table 2), 3 μl ATP (10 mM, NEB), 2.5 μl T4 RNA ligase buffer (NEB), 2 μl T4 RNA ligase 1 (30U/ul, NEB), 1.5 μl water and 15 μl PEG8000 (NEB). The ligation mixture was incubated at 25 °C for 2 h and 16 °C overnight. The ligation product was purified using the Zymo Oligo Clean & Concentrator kit (Zymo Research, D4060) according to the manufacturer’s instructions. The sample was eluted in 20 μl water and kept on ice prior to Bioanalyzer analysis (Agilent, small RNA kit) or used directly in the demethylation step.

The yeast strain containing ChrXVII was grown as described under MNase-Seq. 50-100 μg poly(A)-containing RNA (DNase I treated) was fragmented with NEBNext^® Magnesium RNA Fragmentation Module, as described under RNA-Seq, but scaled appropriately (80 μl fragmentation reaction per 100 μg starting total RNA for 4-5 min). Ethanol precipitated and denatured RNA fragments were dephosphorylated with 2 μl Quick CIP (5 U/μl, NEB) in 15.5 μl reaction volume containing 1.5 μl T4 RNA Ligase buffer (NEB) and 2 μl RNase inhibitor (20 U/μl) for 30 min at 37°C. To the heat-inactivated reaction, 2 μl 5 μM of denatured, pre-adenylated 3’ adapter,³⁷ 1.5 μl T4 RNA Ligase buffer (NEB), 9 μl 50% PEG8000, and 2 μl truncated T4 RNA Ligase 2 (200 U/μl, NEB) were added and the sample was incubated for 2 h at 25°C. Heat-inactivated, resulting products were treated or not treated with mRNA decapping enzyme, MDE (100 U/μl, NEB), by splitting the reaction into two tubes, adding 2.5 μl MDE or water to the tubes, and incubating for 1 h at 37°C. 1 μl of 25 μM denatured 5’ adapter ³⁷ , 0.5 μl 100 mM ATP, and 1 μl T4 RNA Ligase 1 (30 U/μl, NEB) were added to the previously heat-inactivated samples and incubated for 2 h at 25°C. The samples were reverse transcribed, amplified, and purified as described under RNA-Seq. Both MDE treated and control sample were paired-end sequenced with NextSeq Mid and NextSeq High.