Zymo dna clean and concentrator kit

The UEc isolates were evaluated for the absence of papG variants and the presence of the papF gene by PCR using specific primers; additionally, deletion and insertion mutations in the papG locus were evaluated by DNA sequencing (S1 Table). The amplifications were performed with a high-fidelity Pfu polymerase from Thermo-Fisher Scientific (CA, USA), and the products were cleaned and concentrated using a Zymo DNA Clean and Concentrator kit from Zymo Research (CA, USA). The amplicons were cloned into a pJET-Blunt 1.2 vector (Thermo-Fisher Scientific, CA, USA) and were subjected to sequencing on a 3730xl DNA Analyzer from Applied Biosystems^™ (CA, USA) at the “Unidad de Secuenciación-Instituto Nacional de Medicina Genómica” (CDMX, Mexico). The obtained sequences were analyzed using the ClustalO (https://www.ebi.ac.uk/Tools/msa/clustalo/) and Basic Local Alignment Search Tool (BLAST) algorithms from the National Center of Biotechnology Information (NCBI). PCR was used with specific primers to identify the presence of other insertion sequences (IS) in the pap loci and to amplify all pap genes (papI to papG) as well as the following internal regions: papI to papE, papI to papA, and papA to papE.

We employed a long-range PCR (SequalPrep Long PCR Kit, Life Technologies, Grand Island, NY, USA) method to amplify the complete mitochondrial genome. First, we used the following overlapping primers to amplify the mtDNA in two halves: Set 1 (10 Kb amplicon) with forward (3301, GCC AGC CTG ACC CAT AGC CAT AAT AT) and reverse (13367, GAG AGA TTT TAT GGG TGT AAT GCG G); Set 2 (7.5 Kb amplicon) with forward (12791, TCC CAC TCC TAA ACA CAT CC) and reverse (3880, TTT ATG GGG TGA TGT GAG CC). For primer set 1, we used the following PCR conditions: 94 °C for 2 min; 10 cycles: 94 °C for 10 s, 57 °C for 30 s, 68 °C for 10 min; 25 cycles: 94 °C for 10 s, 57 °C for 30 s, 68 °C for 12 min; 72 °C for 5 min (Gene Amp PCR system 9700, Applied Biosystems, Foster City, CA, USA). For primer set 2, we used the following PCR conditions: 94 °C for 2 min; 10 cycles: 94 °C for 10 s, 51 °C for 30 s, 68 °C for 7.5 min; 25 cycles: 94 °C for 10 s, 51 °C for 30 s, 68 °C for 9 min; 72 °C for 5 min. Lastly, we cleaned the PCR products with the Zymo DNA Clean and Concentrator kit (Zymo, Irvine, CA, USA) and pooled the mtDNA for library preparation.

ATAC-seq was performed, in duplicates, as described in^{79 (link)}. Briefly, cells at each time point were dissociated using Accutase, and 50,000 cells were subjected to the tagmentation reaction. Cells were first washed in resuspension buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 3 mM MgCl₂ in water), following which nuclei were isolated in 1 ml lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl₂, 0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin in water) on ice for three min. Nuclei were rinsed once in wash buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, and 0.1% Tween-20) and tagmentation was carried out using 2.5 ul Tn5 transposase (Illumina 15027865) for exactly 30 mins. Following tagmentation, DNA was purified using the Zymo DNA Clean and Concentrator kit (Zymo, D4033). Libraries were prepared using Q5 polymerase and unique indices were added to each sample. First, gap filling was performed at 72 °C for 5 mins followed by five cycles of 98 °C, 20s, 63 °C, 30s, and 72 °C 1 min. After initial amplification, tubes were held on ice, while quantitative PCR was run on 1 µl of the pre-amplified library to determine additional number of cycles needed. Libraries were sequenced on HiSeq2500 using PE50.

ATAC-seq was performed as described previously (Buenrostro et al., 2013 (link); Corces et al., 2017 (link)) Briefly, cells were dissociated and treated with DNaseI (Worthington) and 50,000 viable cells were sorted as DAPI negative. Cells were pelleted at 500 RCF for 5 min at 4°C and resuspended in ATAC-resuspension buffer containing 0.1% NP40, 0.1% Tween20, and 0.01% Digitonin and incubated on ice for 3 minutes. Following wash-out with cold ATAC-Resuspension Buffer (RSB, 10 mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2 in sterile water) containing 0.1% Tween20, cells were pelleted and resuspended in 50 μL transposition mix (25 μL 2x TD buffer, 2.5 μL transposase (100nM final), 16.5 μL PBS, 0.5 μL 1% digitonin, 0.5 μL 10% Tween20, 5 μL H₂O) and incubated for 30 minutes at 37°C with shaking. The reaction was purified using the Zymo DNA Clean and Concentrator Kit, and amplified using PCR primers defined in Buenrostro et al. (2013) (link). Libraries were purified using the Zymo DNA Clean and Concentrator Kit, quantified using the KAPA Library Quantification kit and quality assessed by Bioanalyzer (also used to determine average size for pooling libraries). Samples were sequenced on the HiSeq4000 platform (2x 75bp).

Primers were designed to amplify ~1000 bp of each target genes’ protein coding sequence (S2 Table). Each primer also included 15 bases of the T7 promoter sequence at the 5’ end to be used in a 2-step PCR from wild-type genomic DNA sequence. The PCR product was purified using a Zymo DNA Clean and Concentrator kit (Zymo Research) and used as a template for another round of PCR using primers containing the full-length T7 promoter sequence. After a second purification using a Zymo DNA Clean and Concentrator kit the PCR product was used as a template in a T7 RiboMAX express RNAi System (Promega) following the manufacturer’s protocol. Purified dsRNA was injected at a concentration of 500 ng/μL into L4 or young adult hermaphrodites using a Narishige injection apparatus, a Parker Instruments Picospritzer II, and a Nikon Eclipse TE300 microscope with DIC optics. Excess dsRNA was stored at –80°C. Injected worms were allowed to recover on a seeded NGM plate for 24–36 hours at 20°C before harvesting embryos for imaging.

MethylC-seq libraries were generated using post-bisulfite adapter tagging (PBAT) to avoid the bisulfite-induced loss of intact sequencing templates as described [48 (link)] with the following modifications. Briefly, genomic DNA was subjected to bisulfite treatment for 200 min with EZ DNA Methylation-DirectTM Kit (Zymo D5020). Bisulfite-treated DNA was then preamplified for two cycles with primers (5′-CCCTACACGACGCTCTTCCGATCTNNNNNN-3′) containing random hexamers and purified using the Zymo DNA Clean and Concentrator kit. Adaptor primers (5′-CAGACGTGTGCTCTTCCGATCTNNNNNN-3′) were added to preamplified products and then amplified for 12 PCR cycles with indexing primers for Illumina sequencing. Methylome libraries were purified using Beckman Coulter AMPureXP DNA beads. Libraries quality checked for fragment length between 200 and 600 bp were used for sequenced in single-read mode on an Illumina HiSeq2500 or Nextseq instrument.