GSM4630748: Sample 18_leaf_dcl234_Rep2.sRNA-seq; Arabidopsis thaliana; ncRNA-Seq
samples 1 to 28 : Total RNA extracted with TRIzol reagent (Thermo Fisher Scientific) and size selected for 18-30nt RNA molecules with denaturing urea gel; Samples 29 to 69 : genomic DNA extracted with Quick-DNATM Micro prep Kit (Zymo D3020) Samples 1 to 28 : Size selected RNA was used to synthesise cDNA with NEBNext Multiplex Small RNA Library Prep Set for Illumina kit (cat. no E7300; New England Biolabs) according to the manufacturer's recommendations. Various numbers of PCR cycles were used to amplify cDNAs: 18, 20, 22, and 24 PCR cycles for globular; 14, 16, 18, and 20 PCR cycles for early heart and 3 weeks old leaf samples. Final PCR amplicons were initially run on a 90% (v/v) formamide/8% (w/v) acrylamide gel for 30 min at 5W, followed by 30W for ≥2 h, and stained with SYBR Gold (1:10,000; Thermo Fisher Scientific). Intensity of PCR amplicons between 137-bp and 147-bp  Fluorescence intensities of amplicons were examined across the PCR cycles, 137-bp to 149-bp corresponding to 18- nucleotide to 30- nucleotide sRNAs with adapters respectively was inspected under UV transilluminator. Amplicons with  non-saturated signals from PCR cycles were gel-purified. Gel purified small RNA libraries are resuspenden in 15 μL of Elution Buffer (Qiagen). Finally small RNA libraries quality checked for expected size range with Agilent High sensitivity NGS fragment Kit (DNF-474-1000) were sequenced on a HiSeq 2500 instrument (Illumina) in 50 base single-end mode. Samples 29 to 69 : Genomic DNA subjected to genomic DNA  bisulfite treatment for 200 minutes with EZ DNA Methylation-DirectTM Kit (Zymo D5020).preamplified for two cycles with primers (5′-CCCTACACGACGCTCTTCCGATCTNNNNNN-3′) containing random hexamers and purified using the Zymo DNA Clean and Concentrator kit. Adaptor primers (5′-CAGACGTGTGCTCTTCCGATCTNNNNNN-3′) were added to preamplified products and then amplified for 12 PCR cycles with indexing primers for Illumina sequencing. Methylome libraries were purified using Beckman Coulter AMPureXP DNA beads. samples 1 to 28 : smallRNA-seq; samples 29 to 69: MethylC-Seq
GEO Accession
GSM4630748