Phenol chloroform extraction

Genomic DNA was harvested with protein K buffer (10 mM Tris-HCl at pH 8.0, 100 mM NaCl, 50 mM EDTA, 0.5% SDS, 2 mg/ml protein K), digested with EcoR1, and purified by phenol–chloroform extraction (Invitrogen; cat. no. 15513-039). Three micrograms of DNA from each sample was subjected to the bisulfite reaction according to the protocol of the EpiTect Plus DNA Bisulfite Kit (Qiagen; cat. no. 59124). Fifty nanograms of bisulfate-treated DNA from each cell line was applied to the qPCR or bisulfite sequencing PCR assay. A premixed methylated DNA calibration standard (EpigenDx, Hopkinton, MA, USA; cat. no. 80-8060M-PreMix) was used to measure the methylation intensity of the samples analyzed by qPCR. After the bisulfite sequencing PCR reaction, the PCR products were cloned with a TOPO TA Cloning Kit (Invitrogen; cat. no. 45-0030) for sequencing, and the sequences were analyzed using the BiQ DNA methylation analysis platform at http://biq-analyzer.bioinf.mpi-inf.mpg.de/example.php.
Primer sequences used for methylation-specific real-time PCRPrimers used for bisulfite sequencing

Tissue samples were quick-frozen in liquid nitrogen and stored at −80°C until use. Genomic DNA was extracted by first treating the tissue with proteinase K, and then isolating the DNA by phenol-chloroform extraction (Invitrogen, Carlsbad, CA, USA). The presence of the GFP gene in each sample was analyzed using PCR with 1 µg of genomic DNA for each sample, and 0.1 µg of genomic DNA for the positive and negative controls. The PCR comprised 30 cycles of amplification. The DNA content of each sample analyzed for GFP was normalized to that of the Rattus norvegicus Sry gene, which is the sex-determining region on Y chromosome. The Sry copy number per haploid genome is similar among all male rat cells. The presence of the Sry gene was analyzed using 0.1 µg of genomic DNA in the samples and controls with 28 amplification cycles.

RNA of 10- to 11-h-old RNAi and wild-type embryos was extracted using Trizol (Ambion) according to the manufacturer's protocol, followed by digestion with Turbo DNase (Ambion) and phenol/chloroform extraction (Ambion, pH 6.9). Three biological replicates were sequenced (see supplementary material Table S1 for statistics), with each biological replicate consisting of ∼100 pooled embryos (∼15 µl). The RNAi knockdowns resulted in a transcript reduction of RNA-seq reads of the targeted genes by 80-90%, except for Tc-frizzled2 with 40% reduction. Cuticle analysis of sibling animals confirmed the high penetrance of the RNAi treatments (see Fig. 3 for quality controls). A gene was considered downregulated if the transcript number was reduced by half at a false discovery rate below 0.1 (see MA plots in supplementary material Fig. S3A; datasets in supplementary material Table S2). Principal component analysis (PCA) revealed that the respective treatments cluster (Fig. 3C).

Cyclic tetra-adenylate (cA₄) made per RNA target (0.01, 0.1, 1, 10, 25 or 50 nM) was investigated in a 20 μl reaction volume incubating A26 RNA target or A26 phosphorothioate RNA target (Table 1) with 13.5 μg Sulfolobus solfataricus (Sso)Csm complex (~470 nM carrying A26 CRISPR RNA) in Csx1 buffer containing 20 mM MES pH 5.5, 100 mM K-glutamate, 1 mM DTT and 3 units SUPERase•In Inhibitor supplemented with 1 mM ATP, 5 nM α-³²P-ATP and 2 mM MgCl₂ at 70°C for 2 hr. All samples were deproteinised by phenol-chloroform extraction (Ambion) followed by chloroform (Sigma-Aldrich) extraction prior to separating the cOA products by thin-layer chromatography (TLC). TLC was carried out as previously described (Rouillon et al., 2019 (link)). In brief, 1 μl of radiolabelled cOA product was spotted 1 cm from the bottom of a 20 × 20 cm silica gel TLC plate (Supelco Sigma-Aldrich). The TLC plate was placed in a sealed glass chamber pre-warmed at 37°C containing 0.5 cm of a running buffer composed of 30% H₂O, 70% ethanol and 0.2 M ammonium bicarbonate, pH 9.2. After TLC the plate was air dried and sample migration visualised by phosphor imaging. For analysis, densiometric signals corresponding to cA₄ was quantified as previously described (Rouillon et al., 2019 (link)).