DNA isolation, PCR and Sanger sequencing
Total genomic DNA was isolated using the DNeasy Blood and Tissue Kit
(Qiagen, Hilden, Germany) for Western European samples, and using the
Syngen Tissue DNA Mini Kit (Syngen Biotech, Wrocław, Poland) for
Eastern European samples, according to the manufacturer’s protocols.
Primers (Table S2) were designed based on the Microtus arvalisgenome (NCBI:txid47230, GCA_007455615.1) using Primer-BLAST (NCBI), in
two overlapping fragments, each 1100-1200 bp in length. Using those
primers, the last ~829 bp of intron 8 and the first
~849 bp of exon 9 of the Tshr gene was amplified
by PCR using DreamTaq (ThermoscientificTM, Waltham,
Massachusetts, United States). A mastermix containing: 15.7 μL ultrapure
H2O, 2 μL Dreamtaq Buffer (10x), 0.4 μL dNTP mix (10
mM), 0.4 μL Forward primer (10 μM), 0.4 μL Reverse primer (10 μM), 0.1
μL DreamTaq DNA polymerase (5U/ μL) was prepared for each reaction.
Twenty μL-reactions (1 μL DNA + 19 μL mastermix) were carried out for
each sample by using a thermalcycler (S1000TM,
Bio-Rad, Hercules, California, United States) (Table S3). Following PCR,
an enzymatic clean-up with ExoSAP-IT reagent (Applied
BiosystemsTM, Foster City, California, United Sates)
was performed in order to remove excess primers and nucleotides. Five μL
of cleaned PCR product and 5 μL of the forward or reverse primer (5 μM)
were transferred to a new 1.5 ml tube and sent out for Sanger sequencing
(Eurofins Genomics, Ebersberg, Germany). The intronic fragment was
sequenced in two directions, while the exonic fragment was sequenced in
the forward direction only.