GSM7793898: (6-4)PP XR-seq rep1; Arabidopsis thaliana; OTHER
After 30-min incubation in the dark at 24 °C, the seedlings were frozen with liquid nitrogen, and were ground using mortar and pestle. The resulting powder was resuspended in 400 µl of STES buffer (200 mM Tris⋅HCl pH 8.0, 500 mM NaCl, 0.1% SDS, 10 mM EDTA) and 400 µl of phenol:chloroform (20:1). The sample was homogenized by vortexing with acid-washed glass beads for 30 min at 4 °C, followed by centrifugation at 14,000 rpm for 10 min at room temperature. The supernatant was treated with 10 µl of RNAseA (R4642; Sigma) for 1 h at 37 °C, then with 10 µl of proteinase K (P8107S; NEB) for 1 h at 60 °C. The excision products were obtained by ethanol precipitation and purified by immunoprecipitation with a (6-4)PP-specific antibody obtained from Cosmo Bio. (NMDND001). 5′ and 3′ adapters compatible with the Illumina TruSeq Small RNA protocol were ligated to excision products. Ligation products were immunoprecipitated with (6-4)PP antibodies(CosmoBio USA), and photoreversed with purified photolyase to remove(6-4)PPs interfering with following PCR approaches. Analytical PCR was performed using one percent of the sample to decide the minimum number of cycles required for preparative scale PCR amplification. The repaired ligation products were PCR-amplified using 50- and 63-nt-long primers adding specific barcodes compatible with the Illumina TruSeq Small RNA kit. The correct size PCR products representing the library were gel purified, and then sequenced in the Illumina HiSeq 4000 platform, and single-end 50-nt reads were generated.