Klenow fragment of dna polymerase 1

Tn4001mod on plasmid pISM2062 [28 (link)] was modified by adding a 6xHis-tag and a multiple cloning site: a 51-bp DNA fragment, created by annealing oligonucleotides HisC-f and HisC-r, was inserted between the BamHI and SmaI cleavage sites of pISM2062, resulting in plasmid pTnHis. The M. gallisepticum gene mgc2 was then amplified by PCR using genomic DNA of strain Rlow as template and primers ISM-mgcF and ISM-mgcR, and subcloned into pTnHis using the BamHI and SphI cleavage sites. The resulting plasmid, pTHmgc, was linearized with NotI, treated with the Klenow fragment of DNA polymerase I (New England Biolabs GmbH, Frankfurt/Main, Germany) to fill in the 5′ overhang, and subsequently digested with BamHI. A 1093-bp fragment was gel-purified and ligated to a 3.5-kb fragment of plasmid pINT [27 (link)], obtained after digestion with BamHI and SfoI.

First strand cDNA was synthesized with SuperScript IV reverse transcriptase (ThermoFisher Scientific, Waltham, MA) and random hexamers starting from 200 ng of dsRNA. The second strand DNA was synthesized with Klenow fragment of DNA polymerase I (New England Biolabs, Ipswich, MA). The synthesized dsDNA was precipitated by 100% ethanol and then end-repaired and A-tailed by adding with the EA enzyme provided by the KAPA Hyper Prep kit (KAPA Biosystems, Wilmington, MA). The ligation of the treated dsDNA with adaptor motor mix was performed by the ONT ligation sequencing kit following the ONT protocol SQK-LSK109. The prepared dsDNA was loaded on flow cells and sequencing was performed with MinION for eight hours. Reads obtained from sequencing were real-time analyzed using the ONT EPI2ME WIMP workflow.

Plasmids used in this study are listed in Table 1. The pRFP-C-RS vectors used for transient expression of different shRNAs were purchased from OriGene (TF704233, RefSeq NM_001100975) (Rockville, MD, USA). These vectors express both the red fluorescent protein (RFP) and short hairpin RNAs (shRNAs) that can mediate gene knockdown via the RNAi pathway. To stably express these shRNAs from recombinant lentivirus the shRNA genes were moved from the pRFP-C-RS plasmids into pHAGE vectors [20 (link)] to allow for packaging into lentiviral particles. These plasmids were made in several steps. First, the GFP gene was deleted from the vector pHAGE-Ubc-GIR [20 (link)]by digestion with NotI and NdeI followed by filling of the blunt ends using the Klenow fragment of DNA polymerase I (New England Biolabs, Ipswich, MA, USA) and religation to generate pJS66. The different shRNA genes with their promoters, or the multiple cloning site alone, were then amplified using the polymerase chain reaction (PCR) from the different pRFP-C-RS plasmids using primers, JSO197 (GgactagtGAATTCCCCAGTGGAAAGAC) and JSO198 (GgactagtAAGCTTTTCCAAAAAAGTCTTCT). The PCR products were digested with SpeI and ligated into similarly digested pJS66, creating JS67, JS68, JS69 and JS70, carrying the RFP gene as well as a polylinker, the scrambled shRNA, shRNA3 or shRNA5, respectively, in the pHAGE backbone.