Phusion hs 2

For RNA analysis Hela HA cells were transfected with 1 μg of mneoI/mneoM-tagged reporter construct. RNA was isolated 48 h post-transfection using the peqGOLD Total RNA Kit (VWR). Northern blot analysis (3 μg total RNA) was performed using the NorthernMax - Gly Kit (Thermo Scientific). MneoI/mneoM-tagged RNAs were detected using a biotinylated neo-sense riboprobe; L1-containing transcripts using an antisense riboprobe directed against the 5′ part of ORF2. The Thermo Scientific Biotin Chromogenic Detection Kit was used for detection. For RT-PCR 500 ng RNA were digested with DNAse I (Thermo Scientific). 250 ng of the DNAse digested RNA were subsequently reverse transcribed using the Verso cDNA synthesis kit (Thermo Scientific) and an anchored oligo dT primer containing a PCR primer binding site at its 5′ end (5′-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3′). Resulting cDNAs were amplified using Phusion HSII (Thermo Scientific) and a downstream primer complementary to the 5′ end of the RT primer (PCR anchor; 5′-GACCACGCGTATCGATGTCGAC-3′). H8_43_Kpn (5′- GCGGTACCTATCGAAAGCTGATGAAATGCTC-3′; H8_43_mneoI splice variant detection) and GS87 (5′- GCCATTGAACAAGATGGATTGCACGCAGG-3′; correct mneoM polyadenylation) were used as upstream primers. PCR products were cloned into pJET1.2 (Thermo Scientific) and sequenced.

The rearranged V, D, and J gene segments of the TRB locus were amplified together in a multiplex PCR using TRB-A/-B primer pools and 250 ng of genomic DNA [33 (link)]. The primers were purchased from Metabion International AG (Martinsried, Germany). As described in Schliffke et al. [34 (link)], two consecutive PCR reactions were performed to generate TRB fragments tagged with Illumina-compatible adapters for hybridization to the flow cell and seven nucleotide barcodes for sample identification. All PCRs were performed using Phusion HS II (Thermo Fisher Scientific Inc., Darmstadt, Germany). After gel electrophoretic separation, TRB amplicons were purified using the NucleoSpin^® Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany), quantified on the Qubit platform (QIAGEN, Hilden, Germany), and pooled to a final concentration of 8 nM. The quality of the TRB amplicon pools was controlled on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany) before undergoing NGS. The samples were sequenced with a mean sequencing depth of 80,520 reads (range 42,440–137,852 reads).
NGS and demultiplexing was performed on an Illumina MiSeq sequencer (600-cycle single indexed, paired-end run, V3-chemistry). Analysis of the rearranged TRB loci was computed and plotted as previously described [35 (link), 36 (link)].

Elements were amplified from genomic DNA using primers in the flanking sequence and Phusion HSII (Thermo Scientific). Orangutan DNA was obtained from the Gene Bank of Primates at the German Primate Center. Primer sequences are provided in Additional file 2: Table S6. To facilitate melting of the VNTR, the denaturation time was extended to 30s and 3% DMSO was added to the reaction mix. Amplicons were subcloned into pJET 1.2 (Thermo Scientific) and sequenced. To obtain complete VNTR sequences, subclones containing the VNTR 5′ and 3′ ends, respectively, were generated using SmaI (H8_43) or MscI (OU3, OU4). 5′ primers localized directly upstream of the CT hexameric repeats and 3′ primers designed to exclude the elements’ polyadenylation signals were used for re-amplification. KpnI and NheI recognition sites, respectively, were introduced into the upstream and downstream re-amplification primers. Amplicons were again subcloned into pJET 1.2, sequenced and transferred into pCEP Neo [12 (link)] and pCEP_mneoM via KpnI/NheI. The human SVA H8_43 displays an 11 bp deletion in the SINE-R region when compared to SVA_E and to the subgroup consensus sequences. To obtain a plasmid with a consensus SVA_E SINE-R for cross-species comparison, the missing 11 bp were introduced by site-directed mutagenesis (NEB Q5 kit).