Neb3 buffer

Isotopically labeled glycine (U-¹³C₂, ¹⁵N) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). AICAR, 5-aminoimidazole-4-carboxamide riboside (AICAR) and adenylosuccinic acid (SAMP) were purchased from Toronto Research Chemicals Inc. (North York, Canada). SAICAR, succinylaminoimidazolecarboxamide riboside (SAICAR), succinyladenosine (SAdo), AIR, 5-aminoimidazole riboside (AIr), CAIR, carboxyaminoimidazole riboside (CAIr), and N¹⁰-formyl-tetrahydrofolate (N¹⁰-formyl-THF) were prepared as previously described [11 (link), 14 (link)]. Calf intestinal alkaline phosphatase (CIP) and NEB3 buffer were purchased from New England Biolabs (NEB, Ipswich, MA, USA), and Dulbecco's minimum essential medium (DMEM), F12 nutrient mix, and fetal bovine serum (FBS) were obtained from Life Technologies, ThermoFisher Scientific (MA, USA). Minimum Essential Medium (MEM) was obtained from BioSera (Nuaille, France). All other chemicals were purchased from Sigma-Aldrich (St. Louis, USA).

Two 3′-UTR RNAs [(+ 31)-xrRNA1 and (+ 31)-xrRNA2] were chemically synthesized and purified by the Agilent 1290 Infinity II chromatography system (Agilent Technologies, Santa Clara, CA; see Supplementary Table S5 online). The synthesized RNAs were phosphorylated using T4 Polynucleotide Kinase (Takara) and purified by the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The modified RNAs were incubated at 90 °C for 2 min, followed by 20 °C for 5 min, then held at 4 °C for RNA folding. Xrn1 digestion reactions were conducted with 3–4 µg of the 3′-UTR RNA (~ 100 pmol) and 2 µg of 5′ monophosphorylated 31-mer control RNA (~ 200 pmol) in NEB3 buffer [100 mM NaCl, 50 mM Tris–HCl (pH 7.9), 10 mM MgCl₂, and 1 mM DTT] (New England Biolabs, Ipswich, MA). The RNA mixture was split between two tubes, and two units of Xrn1 (New England Biolabs) were added to one aliquot while the other served as an Xrn1-negative control. Both mixtures were incubated at 37 °C for 2 h, followed by quenching via addition of an equal volume of Novex TBE-Urea Sample Buffer (Invitrogen). The RNA products were analyzed on 15% denaturing PAGE gel and visualized by ethidium bromide staining.

Plasmids were linearised by restriction digest and purified using Monarch PCR and DNA Clean-up Kit (NEB, USA). 3’UTRs were in vitro transcribed from 1 μg of linearised plasmids using MEGAscript T7 Transcription Kit (Invitrogen, USA) according to the manufacturer’s recommendations. RNA was purified by LiCl precipitation and analysed by electrophoresis in a 1.2% denaturing agarose gel. RNA was then refolded in NEB3 buffer by heating at 85 °C for 5 min followed by gradual cooling to 28 °C. The refolded RNA (1 μg) was incubated with 1U XRN1 (NEB, USA) and 10U RppH (NEB, USA) in 20 μL of reaction mixture containing 1x NEB3 buffer (NEB, USA) and 1 u/μL RNasin RNase Inhibitor (Promega, USA). Incubation was performed for 2 h at 28 °C. The reaction was stopped by adding 20 μL of Loading Buffer II (Ambion, USA), heating for 5 min at 85 °C and placing on ice. The entire volume was then loaded into 6% polyacrylamide TBE-Urea gels (Invitrogen, USA), and electrophoresis was performed for 90 min in 1xTBE. Gels were stained with ethidium bromide and documented using an Omnidoc imager (Cleaver Scientific, UK).

Phosphorylated Sf9-Tau was incubated with 10 units of calf intestine alkaline phosphatase (New England Biolabs, catalog no. M0290) per 1 μg of protein for 24 h at 37 °C in NEB-3 buffer (New England Biolabs). The phosphatase was removed by high salt treatment (5 m NaCl at 70 °C), followed by centrifugation and dialysis to PBS for MALDI analysis. Dephosphorylation during TCSPC experiments was achieved by incubation of Sf9-Tau with 10 units/μg protein of Fast-AP (thermosensitive alkaline phosphate from ThermoFisher Scientific catalog no. EF0651) in PBS buffer at 25 °C.

Water, methanol, and acetonitrile, all LC-MS grade, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium hydroxide and acetic acid were also purchased from Sigma-Aldrich. Minimum Essential Medium (MEM) was obtained from BioSera (Nuaille, France). Calf intestinal alkaline phosphatase (CIP) and NEB3 buffer were purchased from New England Biolabs (NEB, Ipswich, MA, USA), and Dulbecco’s minimum essential medium (DMEM), F12 nutrient mix, and fetal bovine serum (FBS) were obtained from Life Technologies, ThermoFisher Scientific (Waltham, MA, USA).

For in vitro tests of sgRNA activities, sgRNAs were generated by PCR amplifying the sgRNA sequences with a primer introducing a T7 promoter upstream of the sgRNA and performing in vitro transcription using MEGAshortscript T7 Transcription Kit (Thermo Fisher). To test the activity of Alu-directed sgRNAs, 1 µg of genomic DNA isolated from human HEK293T cells was incubated with 3 µg of in vitro transcribed sgRNAs and 3 µg of purified Cas9 protein in 20 µl of 1 x NEB3 buffer (New England Biolabs) at 37°C overnight. DNA was visualized by agarose gel electrophoresis in a 1% agarose gel. After digestion, fragmented gDNA was purified using a column purification kit (Zymo) and ligated into SmaI-digested pUC19. The plasmids were transformed into E. coli DH5α and grown on LB agar supplemented with X-gal. Plasmids from white colonies were isolated and the insert ends were sequenced using primers pUC3 and pUC4. Sanger sequencing was performed by GATC Biotech. The activity of L1-directed sgRNAs was tested by digesting 100 ng of a plasmid fragment with 300 ng of purified Cas9 and 300 ng of in vitro transcribed sgRNA in 10 µl of 1 x NEB3 buffer. The DNA substrate was generated by digesting the plasmid containing a full-length L1 retrotransposon (JM101/L1.3) with NotI-HF (New England Biolabs) and isolating the ~3.3 kb fragment by gel extraction.