Miniprep dna

The untagged L1 EN WT protein expression plasmid was generated by restriction digest cloning. The L1 ORF2 consensus sequence was optimized for expression in E. coli and synthesized in pUC57 by GenScript. The sequence corresponding to residues 1-239 was amplified by PCR using Q5 High-Fidelity DNA Polymerase (NEB), cut with restriction enzymes NdeI (NEB) and XhoI (NEB), and ligated into digested pET26b with T4 DNA ligase (NEB). Individual colonies were tested by colony PCR and the insert was confirmed by sequencing following miniprep DNA (Qiagen). Plasmids expressing ORFeus EN domain only were generated by restriction digest cloning. The sequence corresponding to residues 1-239 from pDA007 (EN WT) or from pDA025 (EN H230A) was amplified as described above, cut with BamHI (NEB) and PacI (NEB), and ligated into the digested pDA007 backbone as described above. Inserts were confirmed as described above. Primer sequences used can be found in Supplementary Data 1.

gDNA from a line 6₁ chicken was subjected to PCR using the degenerate primers as described above, the amplicons were purified with the QIAGEN PCR purification kit, concentrated by ethanol precipitation (27 ), and ligated into a pJET vector using the blunt-end protocol described in the CloneJET™ PCR Cloning Kit (Fermentas). By standard procedures (27 ), chemically-competent DH5α E. coli bacteria were transformed, plated on LB agar plates containing 100 μg/ml ampicillin and incubated at 37°C overnight; multiple colonies were picked the next day and overnight cultures grown for miniprep DNA (QIAGEN). Dideoxy-chain termination (Sanger) sequencing on one strand (from T7 primer) was performed (DNA sequencing facility, Department of Biochemistry, University of Cambridge), and sequences were curated using Bioedit [http://www.mbio.ncsu.edu/BioEdit/bioedit.html, (28 )]. Sequence alignments [including ChIR-AB1 sequences from chicken lines M11 and R11 (15 (link))] were made using Clustal (www.ebi.ac.uk/Tools/msa/clustalo/) with phylogenetic trees visualized using dendroscope (http://dendroscope.org/), or by MAFFT and trees displayed using Archaeopteryx.js (https://mafft.cbrc.jp/alignment/server/). The 39 sequences were deposited in GenBank with accession numbers MK605290 to MK605326 (for EM1T7-1 to EM21T7-2).