Platinum superfi dna polymerase

25 mg of frozen liver tissue was lysed to isolate ~25 μg genomic DNA using DNeasy blood & tissue kits (Qiagen). Adaptor oligonucleotides were synthesized by IDT (Supplementary Table 3). Transposon assembly was done by incubating 158ug Tn5 with 1.4 nmol annealed oligo (contains the full-length Illumina forward (i5) adapter, a sample barcode, and UMI)^{40 (link)} at room temperature for 60 min.
For tagmentation, 200 ng of genomic DNA was incubated with 2 μl of assembled transposome at 55° for 7 min, and the product was cleaned up (20 μl) with a Zymo column (Zymo Research, #D4013). Tagmented DNA was used for the 1st PCR using PlatinumTM SuperFi DNA polymerase (Thermo) with i5 primer and gene-specific primers (Supplementary Table 3). Four different libraries were prepared for gDNA from each mouse with different combinations of primers (i5+Locus_F [UDiTaS], i5+Locus_R [UDiTaS], i5+Insert_F [GUIDE-tag] and i5+Insert_R [GUIDE-tag]). The i7 index was added in the 2nd PCR and the PCR product was cleaned up with Ampure XP SPRI beads (Agencourt, 0.9X reaction volume). Completed libraries were quantified by Tapestation and Qubit (Agilent), pooled with equal mole, and sequenced with 150 bp paired-end reads on an Illumina MiniSeq instrument.

For tagmentation, transposome was assembled as previously described^{39 (link)} using purified Tn5 protein and oligonucleotides purchased from IDT. Two hundred nanogram of genomic DNA was incubated with 2ul of assembled transposome at 55 degrees for 7 mins, and the product was cleaned up (20 μl) with a Zymo column (Zymo Research, #D4013). Tagmented DNA was used for the 1st PCR using PlatinumTM SuperFi DNA polymerase (Thermo) with i5 primer and gene-specific primers (Supplementary Table 3). Two different libraries were prepared for gDNA from each mouse with different combinations of primers (i5 + Locus_F [UDiTaS], i5 + Locus_R [UDiTaS]). The i7 index was added in the 2nd PCR and the PCR product was cleaned up with Ampure XP SPRI beads (Agencourt, 0.9X reaction volume). Completed libraries were quantified by Tapestation and Qubit (Agilent), pooled with equal amounts, and sequenced with 150 bp paired-end reads on an Illumina MiniSeq instrument.

To determine the sequence of the vls locus of B. burgdorferi strain K2, the 10910 base pair region encompassing vlsE and silent cassettes vls2-vls16 was amplified using Platinum™ SuperFi™ DNA Polymerase (Thermo Fisher Scientific) and primers YN-LI_266 and YN-LI_267 (Table 2) and then sequenced with a SMRT Cell™ using 10-h data collection (Pacific Biosciences). The resulting reads were subjected to read-of-insert (ROI) analysis using SMRT Link v6.0.0 (Pacific Biosciences), followed by multiple sequence alignment using Geneious Prime 2019.0.4 (https://www.geneious.com), to obtain the final consensus sequence, which was deposited at GenBank under accession number OP620651.

Details of all plasmids used in this study can be found in Table S1. Htr6-EGFP, Htr7-EGFP, Sstr3-EGFP, Sstr5-EGFP, Htr7[TM5-V241Htr6]-EGFP (Chimera N), and the latter’s AQ>FF mutant have been described (25 (link)), as have EGFP-TEV-Stag-TULP3, EGFP-RABL2B, and Flag-RABL2B (31 (link), 34 (link)). Chimeric constructs, internal deletions and missense mutations were generated by overlap extension PCR (25 (link)). Most other constructs were created by PCR-amplifying the region of interest using primers containing restriction enzyme targets, and mutations where needed. Amplifications were performed with Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific), and the resulting PCR products were digested and ligated into desired vectors. Sequences of all plasmids were confirmed by Sanger DNA sequencing (Eurofins Genomics). Primer sequences and PCR conditions are available on request.

Table S1 Plasmids used in this study.

Reverse transcription was performed with the AMV reverse transcriptase (Roche) or SuperScript IV Reverse Transcriptase (Thermo). DNA was amplified with Q5 High-Fidelity DNA Polymerase (New England BioLabs) or Platinum SuperFi DNA polymerase (Thermo). PCR products were purified using the Wizard SV Gel and PCR Clean-Up system (Promega) or Monarch DNA Gel Extraction Kit (New England BioLabs). To map the 5′ end of transcripts (5′ RACE), the RNA adaptor-oligonucleotide dp124 was ligated to total RNA with RNA ligase 1 (New England BioLabs) in the presence of 1 mM ATP and 15% PEG8000 for 4 h (1 h at 25 °C, 1 h at 16 °C, 2 h at 4 °C); RNA was then extracted and reverse-transcribed as described above. Inosines were mapped by primer extension on glyoxal-treated and RNase T1-digested RNA as described previously^{18 (link)}. Detailed experimental procedures are available at https://www.protocols.io/u/matus-valach. Oligonucleotides (purchased from BioCorp and IDT) that were used as primers and adaptors are listed in Supplementary Table S4.

Nested PCR was performed using Platinum SuperFi DNA polymerase (Thermo Fisher Scientific). To amplify exon 1–2 and exon 3 of KLF2 and exon 34 of NOTCH2, the PCR conditions were 10 s at 98 °C (30 s for the first cycle), followed by 20 cycles of 30 s at 58 °C and 1.5 min at 72 °C (5 min for the last cycle) for the first round of PCR and 30 cycles for the second round of PCR. Amplification products were electrophoresed on 2% agarose gel and stained with ethidium bromide. Presence of somatic mutations in the KLF2 (exon 1–3) and in the NOTCH2 (exon 34) were investigated by Sanger sequencing as described^{7 (link), 9 (link), 10 (link)}. The primer sequences used for nested PCR and sequencing analysis are listed in Supplementary Table S2.