Genescan 500 liz standard

Each single-stranded cDNA library from a highly expressed RNA was PCR amplified with Phusion polymerase (NEB) for 15 cycles with two forward primers, a selection primer (containing an RNA-specific sequence and part of the forward Illumina adapter) and a longer primer containing all of the forward Illumina adapter, and a fluorescent reverse primer that binds to the reverse Illumina adapter sequence as part of the ligated ssDNA adapter (Supplementary Table S3, Supplementary Figure S3, Supplementary Methods (step 58)). Moderate to weakly expressed RNAs (RNase P and the btuB riboswitch) were amplified for 15 cycles without the complete forward Illumina adapter primer first, which was then added for a second set of 15 cycles. Libraries that were derived from cultures that contained both sense and antisense plasmids were amplified separately with one selection primer to visually separate the library qualities of the independent priming locations. The fluorescently tagged amplifications were run on an ABI 3730xl Analyzer with GeneScan 500 LIZ standard (Life Technologies) and checked for the correct full-length product (indicating good RT and PCR) and minimal side product formation. See Supplementary Methods (steps 58–67) for further details.

Each single-stranded cDNA library was PCR amplified with Phusion polymerase (NEB) for 15 cycles with two forward primers, a selection primer (containing a sequence specific to the ECK120051404 terminator and part of the forward Illumina adapter) and a longer primer containing all of the forward Illumina adapter, and a fluorescent reverse primer that binds to the reverse Illumina adapter sequence as part of the ligated ssDNA adapter (Supplemental Table S5). The fluorescently tagged amplifications were run on an ABI 3730xl Analyzer with GeneScan 500 LIZ standard (Life Technologies) and checked for the correct full-length product (indicating good RT and PCR) and minimal side product formation.

P. infestans simple sequence repeat (SSR) loci were genotyped using a modified version of the protocol for 12-plex single sequence repeat genotyping as described previously^{34 (link)}. The P. infestans isolates were run alongside a standard 13_A2 sample (2006_3928A). The Qiagen Type-It Microsatellite PCR kit (Qiagen Corporation, Valenica CA) was used for PCR reactions, and sample volumes were modified to run a 12.5 µL reaction by using 6.25 µL 2× Type-It Master Mix, 1.25 µL of a 10× multiplex primer master mix, 4 µL PCR grade water, and 1–2 µL of template DNA (5–10 ng). Thermal cycling conditions as described earlier⁶⁰ . Fragments were analyzed on an Applied Biosystems 3730xl DNA analyzer. The peak size was determined against a GeneScan 500 LIZ standard and alleles were scored manually using Peak Scanner 2 (Applied Biosystems, Foster City, CA), and fragment lengths were rounded to the nearest whole number for analysis.