Illumina HiSeq 2000 sequencing; Bulk segregant sequencing (Bulk-Seq) analysis to identify paternal suppressors of medea in Arabidopsis thaliana
Bulk segregant sequencing (Bulk-Seq) analysis to identify paternal suppressors of medea in Arabidopsis thaliana
Ler and Cvi-0 were reciprocally crossed to generate Ler/Cvi-0 hybrids. These were then used to pollinate homozygous mea/mea or Ler plants. Three independent replicates were generated, each using different parental individuals and at different days. In total, around 10,000 and 4,000 F2 seeds were generated frommea/mea and Ler mothers, respectively. Seeds were surface sterilised for 10 minutes using 1% sodium hypochlorite, washed extensively with water, and sown in MS medium. After 10–14 days of growth at 22°C, leaves from viable seedlings were collected (825, 800, and 775 seedlings for each of theWTpool replicates; 600, 400, and 400 individuals for each of the mea pool replicates). DNA was extracted in groups of 50 leaves using the DNeasy Plant Mini Kit (Qiagen cat. 69104), and quantified using the Qubit dsDNA HS Assay kit (Life Technologies cat. Q32854). DNA from the different extractions was pooled in equi-amounts, precipitated using sodium acetate and isopropanol, and resuspended in 25 μl TE buffer for each replicate at a final concentration of 100–150 ng/μl. The TruSeq DNA Sample Prep Kit v2 (Illumina) was used in the succeeding steps. DNA sam- ples (1 μg) were sonicated and the fragmented DNA samples end-repaired and polyadenylated. TruSeq adapters containing the index for multiplexing were ligated to the fragmented DNA samples. The ligated samples were run on a 2%agarose gel and the desired fragment length was excised (50bp +/- the target fragment length). DNA from the gel was purified with MinE- lute Gel Extraction Kit (Qiagen). Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit (1.0) Fluorometer and the Caliper GX LabChip GX (Caliper Life Sciences). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.
Experimental Factor: Ler x [Ler/Cvi hybrid]
genotype