Mgcl2

Real-time duplex PCR with subsequent High Resolution Melting was performed in a total volume of 25 μL with 1 U FastStartTaq Polymerase (Roche Diagnostics GmbH), 200 μmol/L of each dNTP (Roche Diagnostics GmbH), 0.2 μmol/L of each reverse primer, 0.4 μmol/L of forward primer (S3 Table), 1 x PCR buffer (incl. 2 mmol/L MgCl₂, Roche Diagnostics GmbH), 1 x EvaGreen (Jena Bioscience) and 30–50 ng of template DNA. Amplification and analysis was done in a LightCycler 480 (Roche Diagnostics GmbH) using filter set 465–510 with an initial denaturation at 95°C for 10 min, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s with a single measurement at the end of each cycle and a final elongation at 72°C for 7 min. Subsequently, High Resolution Melting was done at 95°C for 1 min (4.4°C/s), 40°C for 1 min (2.2°C/s), 70°C for 1 s (1°C/s), an increase to 90°C with the continuous acquisition mode (0.02°C/s), and 40°C for 1 s (2.2°C/s). The samples were genotyped by using the Melt Curve Genotyping Analysis Module of the LightCycler 480 Software (release 1.5.0 SP3, Roche Diagnostics GmbH). When calculating the first negative derivative, the wild-type amplicon and the deletion specific amplicon appeared as clearly distinguishable melting peaks with a T_m at 80.5°C and 79.0°C, respectively (S1 Fig).

MALDI-TOF mass spectrometry (Microflex LT, Bruker Daltonik GmbH, Bremen, Germany) was used to verify species identity. Bacterial isolates were differentiated via enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), as described previously [7 (link)]. The total volume per sample was 50 µL with each sample containing GoTaq Flexi Buffer, 4mM MgCl₂, 0.2 mM dNTPs (Roche Applied Science, Penzberg, Germany), 2.0 μM of the forward primer (ERIC-1: 5′-ATGTAAGCTCCTGGGGATTCAC-3′) as well as 2.0 µM of the reverse primer (ERIC-2: 5′-AAGTAAGTGACTGGGGTGAGCG-3′), 2.5 U of GoTaq G2 Flexi DNA Polymerase and approximately 50 ng of template DNA.
The PCR-reaction started with an initial denaturation at 95 °C for 7 min, followed by 35 cycles of denaturation at 94 °C for 30 s. The primer annealing was done at 52 °C for 1 min, the elongation at 72 °C for 8 min and a further, final elongation step at 72 °C for 16 min. Afterwards, the DNA-fragments were run on a 1.5% TBE-agarose gel at 70V for 3h at room temperature and visualized via GelStudio SA System (Analytik Jena, Jena, Germany). Further analyzation of the DNA fingerprints was done using GelQuest and ClusterVis (Sequentix, Klein Raden, Germany).

SNPs within the SORL1 and PPAR G gene were selected according to the following methods: 1) SNPs, which have been reported associations with AD and not been well studied; 2) SNPs, the MAF of which were more than 5%. At last, three SNPs of SORL1 gene and three SNPs of PPAR G gene are selected for genotyping in the study, including: rs709158, rs1805192 and rs10865710 within PPAR G gene, rs1784933, rs3824966 and rs689021 within SORL1 gene. Genomic DNA is extracted from EDTA-treated whole blood, using the DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genotyping methods for PPAR G gene have been described in our previous study [24 (link)]. Genotyping of three SNPs within SORL1 gene were performed using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. PCR primer sequences for each polymorphism were shown in Table 1. The PCR reactions were carried out in a final volume of 25 μl containing: 10 × PCR buffer, 4.5 mM MgCl₂ (Roche, Germany), 0.4 mM of each dNTP (Fermentas, Germany), 10 pmol of each primer, 30 ng template DNA, 1 U Taq DNA polymerase (Roche, Germany) and sterile distilled water up to 25 μl. PCR conditions were: 94 °C for 15min, then 45 cycles of 94 °C for 20 s, 56 °C for 30 s, and 72 °C for 1min, with a final extension at 72 °C for 30min and then reactions held at 4 °C.

To screen for loss of the target gene in KO cell lines, genomic DNA was isolated 10–14 days post transfection with the DNeasy Blood & Tissue Kit (Qiagen). One hundred nanograms of isolated DNA was mixed with 0.2 mM dNTPs, 2 µM forward primer and reverse primer, 1 unit HiFi Polymerase (Roche) and 1× HiFi reaction buffer supplemented with MgCl₂ (Roche), 20 µl total volume. PCR steps were 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 5 s at 60°C, 50 s at 72°C followed by a final elongation step for 7 min at 72°C. Five microlitres of this reaction was run on a 2% agarose gel to check for the presence of the expected product. To test for the presence of the sgRNA template in cells, genomic DNA was isolated from established cell lines or from freshly transfected cultures and analysed by PCR as described above, except that PCR steps were 5 min at 94°C followed by 30 cycles of 20 s at 94°C, 10 s at 65°C, 15 s at 72°C followed by a final elongation step for 7 min at 72°C. Primer sequences are detailed in the electronic supplementary material, file S1.

RNA isolation as described above. PCR was performed using 1.5 μl cDNA, 10x PCR buffer with 20 mM MgCl₂ (Roche), 200 μM dNTPs (Roche), 6 pmol HTTAS_v1 primer, forward: 5′-CAC CGG GGC AAT GAA TGG-3′, reverse: 5′-GTG CGG ATG GCA AGG ACA G-3′, 2 U FastStart Taq DNA Polymerase (Roche), 1 M ethylene glycol (Sigma-Aldrich), and PCR grade water to a final volume of 30 μl. The PCR program started with a 3 min initial denaturation at 95°C, followed by 40 cycles of 10 sec denaturation at 95°C, 10 sec annealing at 60°C, 10 sec elongation at 72°C, after which a final elongation step was performed at 72°C for 7 min.
PCR products were loaded on a 3% TBE agarose gel and bands were extracted using the NucleoSpin Gel & PCR Clean-up kit (Machery Nagel, Düren, Germany). To confirm the sequence of HTTAS, PCR products were cloned into a pGEM-T Easy vector (Promega) and analyzed by Sanger sequencing using a T7-specific forward primer.

Genomic DNA was extracted from laser-captured OPCs, as well as transfected iPSC-OPCs, and bisulfite-converted, using the Zymo Research EZ DNA Methylation-Direct Kit (BaseClear Lab Products). PCR primers were designed using the PyroMark Assay Design 2.0 software (Qiagen, Supplementary Information S2). The assay for MBP was tested for its sensitivity using the EpiTect PCR Control DNA Set (Qiagen). Product amplification was performed using the following reaction mixture: 1 × buffer with 20 mM MgCl₂ (Roche), 10 mM dNTP mix (Roche), 5 μM forward and reverse primers (Metabion AG), 1 U FastStart Taq DNA Polymerase (Roche), bisulfite-converted DNA, and nuclease-free water to a total volume of 25 μl. PCR cycling was performed as follows: initial denaturation for 5 min at 95 °C, 50 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C; final extension for 7 min at 72 °C. PCR amplicons were sequenced using the Pyromark Q48 instrument (Qiagen) with the PyroMark Q48 Advanced CpG Reagents (Qiagen), according to the manufacturer’s protocol and quantified with the Pyromark Q48 Autoprep software.

Genomic DNA was extracted from larvae at 2.5 dpf or caudal fin tissue from adult zebrafish. Tissue was incubated in 25 μl (larvae) or 75 μl (fin tissue) lysis buffer (40 mM NaOH 0.2 mM EDTA) at 95°C for 20 minutes. The lysed samples were diluted 10 times of which 1 μl was incubated together with 0.5 μM of the forward and reverse primer, 100 μM dNTPs (Roche), 0.25 U Taq polymerase (Roche) and 10x PCR buffer + 15 mM MgCl₂ (Roche) in a total volume of 25 μl. Samples were denatured at 94°C for 3 minutes followed by 35 cycles of amplification consisting of 20 seconds at 94°C, 30 seconds at 58°C and 50 seconds at 72°C, and a final primer extension of 5 minutes at 72°C. To screen for genomic lesions, PCR products were sequenced directly. Primer sequences are listed in S1 Table.