Dneasy protocol

Genomic DNA was extracted from approximately 20 mg of caudal fin tissue from 36 or 37 of the 60–100 archived individuals per population according to the QIAGEN DNeasy protocol for animal tissue (optional RNase treatment included), quantified with the PicoGreen dsDNA assay (Invitrogen), and diluted to a standard concentration of 20 ng/μl. Diluted DNA extracts were submitted to the University of Minnesota’s BioMedical Genomics Center in a 96-well format for sample quality assessment and SNP genotyping using the Sequenom MassARRAY® technology. Three multiplex assays (containing 32, 27, and 12 SNPs respectively) were designed using MassARRAY® Designer software. Sampled F. heteroclitus for which DNA was extracted were genotyped with the first two plexes (because all SNP containing genes were represented) following the iPLEX assay protocol. Genotypes for each individual at each locus were called using the Sequenom System Typer Analysis package.

50–200 mg wet weight fresh algal material was transferred to an Eppendorf tube, frozen in liquid nitrogen and ground using Eppendorf grinders with 10 µl saturated ≤106 microns acid washed glass bead solution before proceeding with the Qiagen DNeasy protocol for Genomic DNA purification from cultured animal cells, starting with the proteinase K treatment. 40 µl proteinase K and 200 µl Buffer AL were added to the sample and incubated at 56°C for 30 minutes, before centrifuging for 2 minutes at maximum speed to separate out the beads. 200 µl ethanol was added to the resulting supernatant, vortexed and pipetted onto the spin column, to proceed with the first centrifugation step. For the final step, DNA was eluted using 100 µl water, instead of 200 µl in order to obtain a more concentrated sample.

Cells in a range from 0.7 up to 20 million cells were resuspended in PBS according to the DNeasy protocol (Qiagen). Resuspension was then aliquoted, treated with ProteinaseK, RNaseA and Buffer AL and incubated for lysis and processed for gDNA isolation. The final DNA concentration was assayed using Picogreen reagent.
For NGS library generation, the barcodes were amplified in 8 independent 50 μL PCR reactions using 1 μg of gDNA per reaction with Titanium Taq and Primers #3323 (PEFwdGEX), #3324 (PECellectaA), and #3197–3223 (one of 27 indexing oligos) for 28 cycles. The product was analyzed by agarose gel electrophoresis to check for the expected ~120bp product and purified using the Agencourt. AMPure XP PCR cleanup kit (Beckman Coulter) and the amount of purified product quantified using a Picogreen DNA concentration assay. Barcode representation of each barcode in the 12,998 element shRNA library (Cellecta) was measured by NGS on an Illumina GA2X system. For good representation of each shRNA in the NGS data 12 million raw Illumina sequence reads were required per sample, which averages 2000 reads per shRNA. The deep coverage shRNA libraries used in this work enable high confidence hit calling at the gene level, rather than analysis of individual shRNAs in the data set.