D1000 screentape for tapestation

For analysis of mitochondrial mt-mRNAs via RNAseq, mitochondria were first isolated according to (Minczuk et al., 2011 (link)). Mitochondrial pellets were resuspended in QIAzol (Qiagen) and RNA was extracted using the miRNeasy kit with on column DNase treatment (Qiagen).
Library generation was performed using the TruSeq Stranded Total RNA LT kit (Illumina, UK) according to manufacturer’s instructions. As part of library generation, cytoplasmic and mitochondrial ribosomal RNAs were removed using RiboZero Gold. Quality of libraries was assessed with a D1000 Screentape for TapeStation (Agilent). Libraries were subjected to high-throughput sequencing using the Illumina MiSeq platform.

After enrichment for mitochondria by differential centrifugation in homogenization buffer (10 mM Tris-HCl (pH 7.4), 0.6 M mannitol, 1 mM EDTA), RNA was extracted from NSun3 patient primary fibroblasts and wild-type controls and DNase and Ribo-zero gold (Illumina) treated. Bisulfite conversion of the remaining RNA fraction was performed using the Imprint DNA Modification kit (Sigma). The reaction mixture was incubated for three cycles of 5 min at 90 °C followed by 1 h at 60 °C and then desalted with Micro Bio-spin 6 chromatography columns (Bio-Rad). RNA was desulphonated by adding an equal volume of 1 M Tris (pH 9.0) and incubated for 1 h at 37 °C, followed by ethanol precipitation. About 120 ng of bisulfite-converted RNA was end repaired with T4 PNK (New England Biolabs) and used for library generation, following the manufacturer's protocol (TruSeq Small RNA library preparation kit (Illumina)). Quality and concentration was assessed with a D1000 Screentape for TapeStation (Agilent). Libraries were subjected to high-throughput sequencing using the Illumina MiSeq/HiSeq platform.

Bisulfite conversion of 2 μg DNase treated RNA was performed using the Imprint DNA Modification kit (Sigma). The reaction mixture was incubated for three cycles of 90°C for 5 min and 60°C for 1 h. Following desalting with Micro Bio-spin 6 chromatography columns (Bio-Rad) twice, RNA was desulfonated by adding an equal volume of 1 M Tris (pH 9.0) and incubated for 1 h at 37°C. After ethanol precipitation, reverse transcription was performed with specific primers (Superscript II, Life technologies). After the first stage PCR with overhang primers (Supplementary Table S1), excess primers were removed with Ampure XP beads. An 8 cycles second round PCR was performed with indexed primers (Nextera XT), followed by another clean-up with Ampure XP beads. Quality and concentration were assessed with a D1000 Screentape for TapeStation (Agilent Genomics). Libraries were subjected to high-throughput sequencing using the Illumina MiSeq platform. After quality trimming and 3′ end adaptor clipping with Trim_Galore!, reads longer than 20 nt were aligned to a computationally bisulfite-converted human reference genome (GRCh38) with Bismark (34 (link)).