Hiseq 2500 v4 platform

For transcriptome sequencing transgenic lines were cocultured in shared HM with according controls for at least four weeks in five independent replicates. After sampling, animals were frozen in TRIzol (Thermo Fisher Scientific) at −20 °C until RNA-extraction with the PureLink RNA Mini Kit (Ambion) according to the manufacturer’s protocol. In addition, the optional on-column DNA digestion was performed. The RNA was eluted in 30 µl and checked for sufficient quality. If necessary, the RNA was purified using 1-butanol and diethyl ether^{72 (link)} and frozen at −80 °C until further use. Total RNA sequencing with previous ribosomal depletion was performed for 60 libraries on the Illumina HiSeq2500 v4 platform, with 125 bp paired-end sequencing of 12 libraries per lane. This resulted in 30–40 million reads per sample after quality control. Quality and adapters were trimmed using PRINSEQ-lite 0.20.4^{73 (link)} and Cutadapt 1.13^{64 (link)}. Subsequently, mapping against the newly assembled transcriptome was performed using Bowtie2 2.2.9^{66 (link)}. All downstream analyses were conducted in ‘R’. Differentially expressed contigs were identified with the package DESeq2 1.16.1⁷⁴ after batch correction with SVA^{75 (link)}.

RNA-sequencing was performed using the Illumina HiSeq 2500 V4 platform (Illumina, San Diego, CA, USA) to produce 125 bp paired end reads, as previously described [10 (link)]. In brief, library preparation, including ribosomal RNA depletion using RiboZero Gold, was carried out using Illumina TruSeq V2 reagents. The libraries were sequenced on 3 × 5 lanes of an Illumina HiSeq 2500 instrument.

Genomic DNA from affected children and family members was extracted from peripheral blood using standard methods. Whole exome capture and sequencing was performed as described to a minimum of 30x coverage^{67 (link)}. Exome sequencing for families 8 and 9 was conducted in collaboration with the Regeneron Genetics Center as previously described^{68 (link)}. Briefly, DNA was sheared (Covaris S2), exome capture performed using the Agilent SureSelect v5 enrichment kit according to manufacturer’s instructions, and libraries were sequenced with 125 bp read-pairs using the Illumina HiSeq 2500 V4 platform. All analyses were performed as described^{69 (link)}. Variants were confirmed by bidirectional capillary dye-terminator sequencing and annotated using the reference sequences, GenBank: NM_018121.4, NM_001136123.2 and NM_015110.4. Capillary sequencing was performed in the MRC Human Genetics Unit, Edinburgh, UK, the University of Birmingham, UK, the Bioscientia Institute for Medical Diagnostics, Germany, the Rare Disease Genomics Department, Yokohama City University Hospital, Japan and the Regeneron Genetics Center, Regeneron Pharmaceuticals Inc., USA.

Total RNA was purified using RNAeasy mini kit (Qiagen) per manufacturer's instructions. cDNA library preparation and sequencing were performed by the Genomic Services Laboratory at Hudson Alpha, USA. Briefly, purified total RNAs (RIN score of 7.0 or higher) were prepared for sequencing using the Ovation RNA-Seq System V2 kit (Nugen) followed by RNA-Seq of 100 paired-end reads using the Illumina HiSeq 2500 v4 platform.

Purified genomic DNA was sonicated to 200 base pairs (bp). 5 µg of fragmented genomic DNA was immunoprecipitated with 5 µg of rabbit 6mA antibody (Abcam) or rabbit IgG overnight at 4 °C in a final volume of 500 µl immunoprecipitation buffer (10 mM sodium phosphate, pH 7.0, 140 mM NaCl, 0.05% Triton X-100). The mixture was incubated with 50 µl protein A/G agarose beads for 2 h at 4 °C and then washed three times with 1 ml of immunoprecipitation buffer. The beads were then treated with proteinase K and the methylated DNA was purified by phenol–chloroform extraction followed by ethanol precipitation for library construction. End repair, 3′-adenylation, adaptor ligation, and PCR amplification were performed according to the Illumina TruSeq DNA sample preparation procedures. The libraries were sequenced using an Illumina HiSeq 2500 V4 platform. Of note, the anti-6mA used in this study can efficiently capture either single-strand or double-strand genomic DNA (Supplementary Figure 1b, c)
It is worth to note that both IgG- and input-based controls have been widely used to call peaks in DNA-IP-seq experiments in many studies^{31 (link),32} . In this study, we found that there was no significant bias between two approaches. For example, at the 0.75-h stage, we found that 96% of Input-controlled 6mA peaks were overlapped with IgG-controlled 6mA peaks (Supplementary Figure 1f).