Phusion hf

Genomic DNA and total RNA were prepared from cells using QIAamp DNA Blood kits and RNeasy kits (Qiagen), respectively. RNA was treated on-column with DNAaseI. CDNA was synthesized with First-Strand cDNA Synthesis kit (GE Biosciences). PCR amplification was done using GeneAmp 9700 (Applied Biosystems) and Phusion-HF (NEB) or Platinum Taq polymerase (Invitrogen). PCR primers to amplify cDNA were designed with forward and reverse primers located in distinct exons. Quantitative reverse transcriptase PCR was performed using SYBR green on a BioRad iCycler. Relative gene expression in triplicate wells was quantified using the ∆∆Ct method with TATA binding protein (TBP) as a reference. Automated direct sequencing of PCR products was carried out by the Johns Hopkins DNA Synthesis and Sequencing Facility. Primer sequences for PCR and direct sequencing are available on request.

Guides against RNF121 were designed and ordered as single stranded oligonucleotides from IDT with sequences 5’-GGATCATTGAGAACACGTAT-3’ (Guide 1) and 5’–GTTCCAGAACTCCATAGTAG-3’ (Guide 2). Guides were phosphorylated, annealed, and ligated into BsmBI digested LentiCrisprV2 (a gift from the Feng Zhang lab; Addgene plasmid # 52961) [53 (link)]. To generate the RNF121 overexpression construct, RNF121 cDNA was generated by reverse transcription PCR from total RNA isolated from HEK293 cells (adherent human embryonic kidney 293 cell line obtained from the University of North Carolina Vector Core) using Trizol following manufacturer’s protocol (Invitrogen, Carsbad, CA). RNF121a DNA was amplified by PCR (Phusion HF, NEB, Ipswich, MA) and cloned into pCDNA3.1+ using EcoRI and NotI sites. Catalytic mutants were introduced into this backbone via Site Directed Mutagenesis. pRK5-HA-Ubiquitin-KO was a gift from the Ted Dawson lab (Addgene plasmid # 17603) [33 (link)]. The TTR enhancer and promoter, as well as the ApoE enhancer and hAAT promoters, were isolated from gblocks (IDT) via restriction enzyme digestion, and cloned along with the MVM intron into an AAV GFP expression cassette.

PCR products from pBAD or pet16 cloned constructs were used as templates for NEB PURE or PUREFREX 2.0 in vitro translation reactions. In short, DNA constructs were amplified using Phusion—HF (NEB) kit using T7 forward primer and gene-specific reverse primer. The PCR product were analyzed on agarose gels and purified using the Zymo Clean DNA gel extraction kit. Equal amounts of DNA (50–150 ng) were used in in vitro reactions. If noted PCR products were used to synthesize RNA using T7 polymerase kit (NEB), purified using NEB RNA purification kit and equal amount of purified RNA was used for in vitro reaction (1–3 µg). In vitro protein synthesis was conducted for 2.5 h at 37 °C if not noted differently. In the case of fluorescent proteins translation was followed in parallel by fluorescence reading using for 2.5 h in 1 min intervals. Same amount of samples were loaded on SDS PAGE gels and western blot analyses were performed as described for in vivo expression experiments.

cDNA template was amplified using primers to the region of interest: 5′-CGAGGCTCTTATGGAACTGATGAATC-3′ (forward) and 5′-GATCCATCGTGCTTTCAGACACATC-3′ (reverse). No amplification was observed in the no RT condition. PCR conditions were: 500 nM primers, 3% DMSO, 1x Phusion HF (NEB #M0531L), 0.5 µL cDNA template, and cycling at (98 °C, 30 s), (98 °C, 10 s; 60 °C, 30 s; 72 °C, 45 s)x35, (72 °C, 7 m), (4 °C, ∞). The additional reverse primer 5′-AAATGCTTCATTGTTACCGTCAGCT-3′ and the additional forward primers 5′-TCCAAAAGAAACGATGTGAGTATGCAG-3′, 5′-CTCTCTTCAGCCAGTTCTTCAGGAT-3′, 5′-ACAATTTCATAAAATGGCTGGCCGA-3′, 5′-CGAGGCTCTTATGGAACTGATGAATC-3′, 5′-GTGCTTTCTCCATTTGCTCTTCCTT-3′ were used for sequencing, along with the primers used for amplification from cDNA. Chromatograms were quantified using ab1PeakReporter (Thermo Fisher).

DNA fragments were amplified by PCR with exTaq-HS (Takara Bio) and Phusion HF (New England Biolabs) DNA polymerases from Ciona genomic DNA or cDNA. The primers that we used were summarized in Appendix Table S1. The amplicons were subcloned into TOPO vectors (life technologies). DIG or fluorescein-labeled RNA probes were synthesized by T7 and sp6 RNA polymerases (Roche) from template DNA plasmid digested by NotI or SpeI (New England Biolabs), and were cleaned by RNeasy mini kit (QIAGEN). We followed the protocol for in situ hybridization described before (Christiaen et al, 2009b (link); Ohta and Satou, 2013 (link)). We detected fluorescein and DIG probes using TSA plus (Perkin Elmer), green (FP1168), and red (FP1170), respectively. Primer sequences are provided in Appendix Table S1.
We used an antibody for phospho histone 3 (pH3) that was previously reported (Shirae-Kurabayashi et al, 2006 (link)) (pH3-ser10-6g3-mouse-mAb, Cell Signaling; #9706; 1:500 diluted). The primary antibody was added together with anti-DIG antibody during FISH process, and secondary antibody (anti-mouse-Alexa-555, Thermo Scientific, A-21127) was used to detect the anti-pH3 antibody after detection of ISH probe by using TSA plus (Perkin Elmer) green (FP1168).

A cassette containing the intron in which each snoRNA resides, and its two flanking exons (For ACA21, RPL23 exons 2–3, for ACA61, SNHG12 exons 4–5) was amplified from genomic DNA using Phusion HF (NEB). The fragment was cloned to pmTurquoise2-H2A plasmid (41 (link)) between the BglII and HindIII sites. A mixture of the two snoRNA expression plasmids was transfected in equal amounts to the pool of variants. As a control, an empty pmTurquoise2-H2A plasmid was co-transfected with the pool of variants. ACA21 and ACA61 were chosen as they complied with the following experimental demands: first, they are expressed in WT HEK-293T cells, and are among the 10 H/ACA snoRNAs with the highest rank of expression in ENCODE cell lines (21 (link)). Second, their rRNA targets have high Ψ levels as previously measured by Ψ-seq (data not shown). Third, they are the sole targeting snoRNAs of their rRNA targets, ensuring potential Ψ signals are not a result of redundancy in targeting by additional snoRNAs. Fourth, they are not part of a cluster of highly similar snoRNAs, thus do not resemble any other snoRNA by >80% (21 (link)). Last, the length of the region encompassing the snoRNAs, the intron they are embedded in and the two flanking exons was <500 nt-long, facilitating the amplification and cloning of the fragment.