Quickchange 2 xl kit

In order to control expression of dCas9-SunTag and scFv-DNMT3A, we acquired doxycycline-inducible open-reading frame expressing vector Pinducer 20 (P20) (Addgene 44,012) from the Thomas F. Westbrook lab and we further exchanged the selection marker of the original P20 vector from neomycin to blasticidin (P20-BSD). The sequence of dCas9-SunTag-2A-BFP and scFv-sfGFP-DNMT3A was then gateway cloned to P20 and P20-BSD, respectively. Catalytic inactive mutation (E756A) of DNMT3A was generated using agilent QuickChange II XL kit based on manufacturer’s instructions in PDONR223-scFv-sgGFP-DNMT3A and then gateway cloned to the P20-BSD vector.

Site-directed mutations of the phosphorylation sites were created using similar methods as the Quickchange^® II XL kit from Agilent technologies by using the primers listed in Table 1 to recreate the whole vector via polymerase chain reaction (PCR) with the mutation in place. The primers with either aspartic acid or alanine substitutions were used to amplify the mutated version of THRUMIN1 using the Gateway vector pBSDONR P1P4 backbone (Qi et al., 2012 (link)). Error-free sequences were recombined into the pEG100 plant expression vector (Earley et al., 2006 (link)) with a pBSDONR P4rP2 YFP clone using LR Clonase II (Invitrogen, Carlsbad, CA, USA) to create a final destination vector to be transformed into Agrobacterium strain GV3101. Internal deletion constructs were created using primers that extend away from the deletion site toward the beginning and end of the gene to create two PCR fragments which were later fused together through PCR extension (Atanassov et al., 2009 (link)). The fused products were recombined into pEG100 in the same manner as described earlier. The mutant genes were then transformed into Arabidopsis Col-0 and thrumin1-2 (SALK_027277) backgrounds using the Agrobacterium-mediated floral dip transformation method (Clough and Bent, 1998 (link)).

To confirm the potential role of TAPT1 deep intronic mutation (c.1237‐52 G>A) in splicing defect, an in vitro minigene assay was done using pSPL3 exon trapping vector (Westin et al, 2021 (link); Iturrate et al, 2022 (link); Rodriguez‐Muñoz et al, 2022 (link)). A genomic DNA fragment from patient cells (V.1(F1)) containing TAPT1 exon 12 flanked by 200 bps upstream and 500 bps downstream intronic sequences were cloned into pSPL3 vector (Invitrogen) using EcoR1/BamH1 restriction sites. Subsequently, the mutant construct was used as a template to generate a rescue construct by introducing c.1237‐52 A>G change using QuickChange II XL kit (Agilent). Both mutant and rescue constructs were verified by direct Sanger sequencing. Then, HEK293T cells were transfected with 4 μg of DNA (pSPL3‐c.1237‐52 G>A, pSPL3‐rescue or empty pSPL3 as a control) using Opti‐MEM (Gibco) and Lipofectamine 3000 reagent (Invitrogen). Total RNA was extracted 24 h after transfection by NucleoSpin RNA kit and 3 μg of RNA was used for cDNA synthesis (Qiagen). To compare the splicing patterns of cells transfected with different constructs, RT–PCR was performed using vector‐specific primers (SD6 and SA2). The PCR products were loaded on 2% agarose gel and purified after gel extraction. The transcripts were analyzed by direct Sanger sequencing. Primer sequences are shown in Table EV1.

Targeted mutations of Bdnf promoter construct plasmids were performed using the Quickchange II XL kit (Agilent Technologies, Les Ulis, France) following the manufacturer instructions. Briefly, 10 ng of SP4 luciferase plasmid was amplified by PCR with sense and antisense mutated primers for 18 cycles. PCR reactions were transformed in XL1 blue ultracompetent E. Coli strain (Agilent, les Ulis, France) on LB Agar Ampicillin petri dishes. Bacterial colonies were picked up. Plasmid DNA were extracted and sequenced by Eurofins (Ivry sur Seine, France) to check for the introduction of the mutation and sequence integrity. Primers for mutagenesis are available in Additional file 1: Table S1. Plasmids were transfected in N2A cells as stated for the luciferase assay.

Recombinant C1qCLR_nc2 (WT and mutants) was produced and purified as previously reported [21 (link)] with the difference that L-ascorbic acid 2-phosphate was added during the production step in the EXPI culture medium at a final concentration of 450 µM and the concentration of L-ascorbic acid was reduced to 250 µM (instead of 568 µM in Fouët et al. 2020a). Previous reports showed that L-ascorbic acid 2-phosphate is more stable than L-ascorbic acid and enhances production yields for some collagen types [43 (link)]. The DNA sequences corresponding to the GPP triplets present between the C1qCLR and nc2 sequences were removed by site-directed mutagenesis with the Quickchange II XL kit (Agilent technologies (Les Ullis, France).