title: GSM3228472: Control m6A Supernatant Rep 1; Arabidopsis thaliana; RIP-Seq
library_construction_protocol: Following control or salt treatment, rosette leaves were collected 4 hrs after first light. Leaves were immediately placed on liquid N2 and ground using a mortar and pestle. RNA was extracted using Qiazol (Qiagen) according to manufacturers instructions. 2 rounds of PolyA+ selection was performed using the Dynabeads mRNADirect kit (Thermo). miCLIP-seq was performed using 3 μg of polyA+ selected mRNA per replicate. Samples were placed at 75 degrees Celsius for 5 minutes and snap cooled on ice for 2 minutes. Samples were brought to 686 uxL with nuclease free water. Next, 10 uxL RNaseOUT, 200 uxL 5X IP buffer (250 mM tris HCl, 750 mM NaCl, 0.5% vol/vol Igepal[CA-6300]), and 3 uxL of m6A antibody (Abcam ab151230) were added to samples which were rotated at 4 °C for 2 hours. While rotating, Protein A bead slurry was washed twice with 1mL 1X IP buffer and resuspended in 1 mL 1X IP buffer, which was supplemented with 0.5 mg/mL BSA and rotated for 2 hours at 4 °C. After 2 hours, RNA was transferred into 3 cm cell culture dish and cross linked twice with 0.15 J/cm2 UV light in an agilent stratalinker. Protein A beads were then pelleted using magnetic strand and washed twice using 1 mL 1X IP buffer. 250 uxL of bead mixture and RNA samples were placed into a 2 mL tube and rotated for 2 hours at 4 °C. After 2 hours, beads were pelleted using a magnetic stand, supernatant was removed and stored at -80 °C as unbound supernatant. Bead bound samples were removed from beads by adding 95 uxL proteinase K buffer, 5 uxL of proteinase K, and treated for 45 minutes at 50 °C, agitating every ten minutes. Supernatant was removed and stored at -80 °C as the m6A+ sample. Beads were washed twice using 300 uxL 1X IP buffer. Supernatant from both washes were also stored as m6A+ samples. All samples were kept at -80°C overnight and precipitated using NaOAc. m6A+ samples were then pooled after resuspension in nuclease free water. m6A+ and – were then prepared into libraries using the strand-specific sequencing library preparation as previously described (Silverman et al, 2014). Strand specific libraries were constructed as previously described (Silverman et al, 2014).
attributes.tag: GEO Accession
attributes.value: GSM3228472