Jm109 high efficiency competent cells

RNA extraction, entire NS5A gene reverse transcription, amplification, cloning and sequencing are explained in detail elsewhere [35 (link)]. Briefly, HCV RNA was extracted from 200 μL of each serum sample using the EasyMag automated extraction system (BioMérieux, Craponne, France). A full-length NS5A gene amplification was performed using outer primers E1 and E2 for the RT-PCR (1665 bp) and inner primers I3 and I41b (1344 bp) for the nested PCR. NS5A PCR products were subjected to direct sequencing using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on the automated ABI3130xl, with the primers used previously in the nested-PCR rounds. NS5A PCR products were also cloned using the p GEM-T Easy Vector, the T4 DNA ligase and JM109 High Efficiency Competent Cells (Promega) following the manufacturer’s instructions. Clones of interest, i.e., bearing the NS5A gene, were then sequenced as described above.
We sequenced a mean of 30 clones per sample and obtained 701 direct NS5A sequences and 1000 clonal NS5A sequences that were added to the previous sequence batches for analyses (37 direct NS5A sequences and 286 clonal NS5A sequences respectively).

Bisulfite conversion was performed on 2 μg genomic DNA using EZ DNA Methylation kit (ZYMO Research, Irvine, CA, USA). The bisulfite converted DNA was amplified by PCR that targeting the TSLPv2 promoter region. Primer sequences for: TSLPv2 CpG forward 5′-TTA GGT ATT TTG GAG AGG GAG TAT TT-3″ and TSLPv2 CpG reverse 5′-AAA TCA AAA TTA AAT AAA ACA AAA AAA A-3′. Target PCR products were purified, cloned to pGEM-T easy vector, and transformed to JM109 High Efficiency Competent Cells (Promega, Madison, WI, USA). Ten clones from each cell line were isolated and sequenced. The percentage of methylation at each CpG dinucleotide (total 29 CpG dinucleotides) in the CpG islands was analysed.

Crispant embryos would be expected to be mosaic. To confirm that the mutation had occurred and to get a rate of mutagenesis, we cloned the heterogeneous PCR products from individual embryos into a vector so that individual mutations could be sequenced. The PCR products were ligated into the pGEM-T easy vector (Promega) according to the manufacturer’s instructions. JM109 High Efficiency Competent Cells (Promega) were transformed with the ligated plasmid and plated on ampicillin resistant agar plates. Plates were incubated at 37°C overnight. Colony PCR was then performed on plasmids with standard pUC/M13 primers (Eurofins, Table S1). PCR products were sequenced subsequently.

The sequences of differentially-expressed probes of the Affymetrix Ovine Gene 1.1 ST Array (http://www.affymetrix.com/analysis/index.affx) were used to obtain full exon sequences using NCBI-BLAST against the sheep genome assembly, Oar v3.1 (http://www.livestockgenomics.csiro.au/sheep/oar3.1.php/). Exon-specific primers were selected using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and reanalysed using Net Primer (www.premierbiosoft.com/netprimer/). Primers used for RT-PCR and RT-qPCR are shown in Table S2A. The location of the primers and their relationship to the locations of the Affymetrix Ovine Gene 1.1 ST whole-genome array probe sets is shown in Table S3. RT-PCR used the FastStart Taq DNA Polymerase Kit (Roche) following the manufacturer’s protocol. PCR products were analyzed by agarose gel electrophoresis, visualized by GelRed/UV transillumination, purified using MinElute PCR Purification Kit (Qiagen), ligated into pGEM-T Easy vector (Promega) and transformed into JM109High Efficiency Competent Cells (Promega). A random selection of color-screened clones were sequenced (Edinburgh Genomics; https://genomics.ed.ac.uk/) with SP6 and T7 primers using the BigDye^® Terminator v3.1Cycle Sequencing Kit (Applied Biosystems, UK).

In order to detect other possible bacterial symbionts, the primer pairs 61F and 1227R, and 10F and 1507R [41 (link)] were used to amplify eubacterial 16S rDNA in individuals from the Chilean and two Australian (Richmond, Canberra) populations (Additional file 3: Table S3). Products obtained from these PCR reactions were cloned and sequenced. Column purified PCR products were ligated into pGEM-T Easy Vector (Promega) followed by transformation of JM109 High Efficiency Competent Cells (Promega) according to the manufacturer’s protocol. White colonies containing inserts were picked, transferred into 20 μl colourless 5× GoTaq reaction buffer (Promega), boiled at 98°C for 10 min, and 1 μl was used as template in colony PCR reactions using the SP6 and T7 promoter primers [50 (link)]. Colony PCR products were treated with 2 μl of ExoSAP mixture containing 0.5u Exonuclease I (New England Biolabs, Ipswich, MA) and 0.25u Shrimp Alkaline Phosphatase (Promega), then incubated at 37°C for 30 min, followed by 95°C for 5 min [50 (link)]. The PCR products were then sent to Macrogen Korea for sequencing. PCR conditions, the number of sequenced clones, GenBank accession numbers and related information are included in Additional file 4: Table S4, Additional file 5: Table S5, Additional file 6: Table S6 and Additional file 7: Table S7.