title: GSM1942138: met1_epiRIL_01_RNAseq_2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Tissue was flash frozen in liquid nitrogen for all experiments including ChIP-seq, RNA-seq and MethylC-seq. DNA was isolated using a Qiagen Plant DNeasy kit (Qiagen, Valencia, CA) following the manufacturer’s recommendations. RNA was isolated using TRIzol (Thermo Scientific, Waltham, MA) following the manufacturer’s instructions MethylC-seq libraries were constructed using the MethylC-seq protocol (Urich et al. 2015). RNA-seq: RNA-seq libraries were constructed using Illumina TruSeq Stranded RNA LT Kit (Illumina, San Diego, CA) following the manufacturer’s instructions with limited modifications. The starting quantity of total RNA was adjusted to 1.3 µg, and all volumes were reduced to a third of the described quantity. CHIP-seq: Immunoprecipitated DNA was end repaired using the End-It DNA Repair Kit (Epicentre, Madison, WI) according to the manufacturer’s instructions. DNA was purified using Sera-Mag (Thermo Scientific, Waltham, MA) at a 1:1 DNA to beads ratio. The reaction was then incubated for 10 minutes at room temperature, placed on a magnet to immobilize the beads, and the supernatant was removed. The samples were washed two times with 500µL of 80% ethanol, air dried at 37C and then resuspended in 50ul of 10 mM Tris-Cl pH8.0. Finally, the samples were incubated at room temperature for 10 minutes, placed on the magnet, and the supernatant was transferred to a new tube, which contained reagents for “A-tailing.” A-tailing reactions were performed at 37C according to the manufacturers instructions (New England Biolabs, Ipswich, MA). The samples were cleaned using Sera-Mag beads as previously described. Next, adapter ligation was performed using Illumina Truseq Universal Y-adapters and T4 DNA ligase (New England Biolabs, Ipswich, MA) overnight at 16C. A double clean-up using the Sera-Mag beads was performed to remove any adapter-adapter dimers and the elution was used for 15 rounds of PCR. Lastly, samples were cleaned up one final time using the procedures described above.
attributes.tag: GEO Accession
attributes.value: GSM1942138