To ensure high stringency, membranes were washed twice for 15 min in 2× SSC and 0.1% SDS buffer and twice in 0.1× SSC and 0.1% SDS buffer at 68°C.
A threshold of 10-15 was used to ensure the stringency of locating matching genes.
For negative controls, ;;UAS-unfF1 virgins were routinely crossed to ;;;OK107-GAL4/+ or ;FRTG13UAS-mCD8/+;;OK107-GAL4/+ males at 22° to continuously monitor and ensure the stringency of the screen.
Slide hybridization and washing protocols were performed according to the manufacturer's instructions, except that hybridization was conducted in the presence of 37.5% formamide at 68°C to ensure proper stringency due to the high G + C content of the H. salinarum genome (67%, [ 32]).
To ensure high stringency, the temperature of hybridization and the post-hybridization washes were estimated for each oligonucleotide probe according to the Tm and adhering to the GC content rule: 30.7°C for AATAC3, 36.2°C for ACT5, AAG5 and AAC5, 38.9°C for AAGAG3, AACAC3 and AAGAC3, 44°C for GATA5, and 54.3°C for AG10, AC10 and GACA5.
We are also providing data to the reviewers from additional control experiments, which we performed with TCR negative hybridomas, in order to ensure that the stringency employed in Figure 4 provides resolving power equal to Figure 3.
SNP calling was performed at very high stringency to ensure accuracy.
Searches were carried out at a stringency of 1e-10 (this relatively low stringency was to ensure that we identified as many immune homologies as possible).
This stringency ensured the development of robust SSR markers in mulberry which can be effectively used for diversity analysis as well as for constructing genetic linkage maps.
