Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Comparison of mouse and worm m6A distribution: PolyA+ transcripts were purified from 75 µg of total RNA using the Dynabeads mRNA purification kit (Life Technologies; cat. no 61006). An equal amount (in µg) of total or polyA+ RNA from adult mouse testes and Bombyx mori BmN4-SID1 insect cell line were mixed together with worm polyA+ or total RNA prior to subsequent processing. In this mixed sample, the mouse RNA serves as an internal control for efficient m6A immunoprecipitation via the unambiguous detection of m6A peaks that are already reported. Fragmentation for total or Poly(A)+ RNA mixture from mouse, worm and BmN4-SID1 was performed as described (Wojtas, M. N. et al.,Mol. Cell 68, 374–387 (2017). For total RNA fragmentation; 5µg of total RNA from mouse testis, adult worms and BmN4-SID1 cells each were used. For polyA+ RNA fragmentation, 2µg of poly(A)+ selected RNA from mouse testis, adult worms and BmN4-SID1 cells each was fragmented. Denaturing urea-PAGE confirmed that the majority of the RNA fragments were in size range of 20-80 nucleotides. A small portion (10%) of fragmented RNA was kept aside to be used as the input sample, while the remainder was subjected to immunoprecipitation. The m6A immunoprecipitation was performed as described (Wojtas, M. N. et al.,Mol. Cell 68, 374–387 (2017). Comparison of m6A distribution between mett-10 KO and mett-10 WT worms: Total RNA was isolated from adult wildtype or Mettl16 (Mett-10) mutant worms (C. elegans), grown on different media, using the Trizol reagent (ThermoFisher Scientific; 15596026). Poly(A)+ transcripts were Comparison of m6A distribution between mett-10 KO and mett-10 WT worms: purified from 75 µg of total RNA using the Dynabeads mRNA purification kit (Life Technologies; cat. no 61006). Poly(A)+ RNAs (2.50 µg) fragmentation and m6A-IP were carried out as described before (Wojtas, M. N. et al.,Mol. Cell 68, 374–387 (2017). Strand specific RNA libraries from the input and the m6A IP RNA were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB; catalogue No. E7560L) following the manufacturer's instructions. The libraries were resolved on 3% high-resolution MethaPhor agarose (Lonza; catalog. No. 50180) gels in 1X TAE buffer at 70 V. Fragments in the size-range of ~150-250 bp were gel-extracted with the use of MinElute Gel Extraction Kit (Qiagen; cat No. 28604). Multiple libraries with different barcodes (at 3′ end) were mixed in equimolar ratios and sequenced in EMBL Gene Core facility, Heidelberg or by iGE3 Genomics Platform, University of Geneva.