Calf intestinal phosphatase

Microsatellite DNA was isolated as described previously [21 (link)]. The extracted gDNA (1 μg) was digested with RsaI (New England Biolabs, Ipswich, MA, USA) for 10 s according to the manufacturer’s instructions. The resulting DNA fragments were treated with mung bean nuclease (New England Biolabs) for 30 min to obtain blunt ends and dephosphorylated using calf intestinal phosphatase (New England Biolabs). DNA fragments of 200–800 bp were separated using electrophoresis on 1.5% agarose gels and recovered using a QIAquick gel extraction kit (Qiagen, Germantown, MD, USA). The recovered DNA fragments were ligated to adapters (SNX/SNX reverse linker) by combining with 60 μM SNX adapter, 5 μL of NEB #2 buffer, 0.5 μL of 100× BSA, 1 μL each of NheI (New England Biolabs) and XmnI (New England Biolabs), 50 mM rATP (Promega, Madison, WI, USA), and 2000 units of ligase (New England Biolabs) in a total volume of 50 μL.

Methylated RNA was purified using the RNA Clean & Concentrator Kit (Zymo Research). The purified RNA was subsequently enzymatically digested to individual mononucleosides using nuclease P1 (Sigma Aldrich), snake venom phosphodiesterase (Sigma Aldrich), and Calf Intestinal Phosphatase (NEB) [2 (link)–4 (link),20 (link),51 (link)]. The digested samples were separated by analytical HPLC using a Luna C18 reverse-phase column (10 μm, 4.6 mm x 250 mm) (Phenomenex) and as in a previously described protocol [20 (link)]. Synthetic 2-methyladenosine and the unmodified mononucleosides were detected by UV absorption at 256 nm, whereas the ¹⁴C-labeled methyladenosines were detected by radiomatic flow scintillation analyzer (Packard 515TR flow scintillation analyzer; Perkin-Elmer).

The genomic DNA of M. abscessus was isolated from a log phase culture using the CTAB method [23 (link)]. The DNA was digested with Sau3A (NEB, Ipswich, MA, USA) and run on 1.5% agarose gel. DNA fragments ranging from 200 to 1500 bp in size were eluted using a gel elution kit (Qiagen, Valencia, CA, USA). The eluted DNA was ligated into the pJEM11 vector, predigested with BamH1, and dephosphorylated using calf intestinal phosphatase (NEB, Ipswich, MA, USA) with an insert-to-vector ratio of 2:1. A ligation mixture was used to transform the chemically competent E. coli DH5α cells and the selected transformants on a Luria Bertani (LB) agar plate containing kanamycin (50 μg/mL).

The loxp-DsRed-loxp-eGFP sequence was PCR amplified from the plasmid pMSCV-loxp-DsRed-loxp-eGFP-Puro-WPRE (#32702, Addgene) by using primers containing Sal1 and Not1 restriction enzyme sites. The amplified PCR fragment was digested with Sal1 and Not1, treated with Calf intestinal phosphatase (New England BioLabs), and agarose-gel purified. The fragmented DNA was ligated into the pENTRA1 vector cut with Sal1 and Not1. The Gateway System^TM (Invitrogen) was used to recombine the pENTR1A shuttle vector with pLenti CMV/TO Zeo DEST (644-1) (#17294, Addgene) generating lentiviral vector 1.
The sequence GTGTACGTG (1HRE) spaced with random 5 base pairs of nucleotides was used to generate tandem copies from 1 to 9 HREs that were directly synthesized by Integrated DNA Technologies (IDT) as gBlocks (Coralville). The TK minimal promoter followed by 770-bps β-globin intron sequence as well as a CRE and CRE-ODD nucleotide sequence were also independently synthesized as gBlocks (IDT). The pRRLSIN.cPPT.PGK-GFP.WPRE (#12252, Addgene) was digested with ECORV and BAMH1 to remove the PGK promoter and EGFP cassette. In-Fusion cloning (Clonetech) was used to ligate the HRE gblock (s), the β-globin gblock, and the CRE or CRE-ODD gblock into the linearized pRRLSIN.cPPT.PGK-GFP.WPRE vector to generate the vectors shown in Supplementary Fig. 1a.