Hs ngs kit

Total RNA was individually extracted from control and heat-treated immature grains using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, Inc., Spain) and following manufacturer’s instructions. For the RNA sequencing, three biological replicates were analyzed per condition and genotype, each composed of a pool of three immature grains (total 100 ng of RNA). Both library preparation and sequencing were performed and optimized by the Genomics Unit of the Instituto Gulbenkian Ciência, Oeiras. mRNA-libraries were prepared using the SMART-seq2 protocol adapted from Macaulay et al. (2016) (link) Illumina^® libraries performed used the Nextera protocol adapted from Baym et al. (2015) (link). The libraries quantification and quality verification were done using the Agilent Fragment Analyzer in combination with HS NGS Kit (Agilent Technologies, Santa Clara, California). Libraries were sequenced in the NextSeq500 Illumina^® Sequencer using 75 SE high throughput kit (Illumina, San Diego, California) and 937302653 reads were obtained from the 24 samples.

Total DNA was extracted from individual samples using the ISOLATE Fecal DNA kit (Bioline, London, UK) following the manufacturer protocol, and their concentrations were quantified using Qubit fluorometric quantitation (ThermoFisher, Foster City, CA, USA). For cost-effective reasons and given that our study seeks to describe the bacterial composition of each lizard population as a whole, samples from each island/islet with matching sex, season, and collecting year were pooled in equimolar concentrations, obtaining a final volume of 30 μl at 20 ng/μl per sample (see Table 1 for details on the number of faecal samples pooled per location). A total of 48 samples were submitted to the Roy J. Carver Biotechnology Center (University of Illinois, USA) for amplification of the V4 region of 16S rRNA in a microfluidic high-throughput multiplexed PCR platform (Fluidigm). The primer set 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [28 (link)] were used, and CS1 and CS2 Fluidigm universal tags, barcode labels specific to each sample and Illumina adapters i5 and i7. The resulting amplicons were validated on a fragment analyzer (Agilent) using the HS NGS kit (DNF-474–33). Sequencing was conducted on an Illumina MiSeq v2 platform yielding 2 × 250 bp paired-end reads.