alias: GSM3681158
title: GSM3681158: rz−1b rz−1c replicate 2 mutant-1-2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: CB-RNA-seq : Nuclei from 2g of grinded powder of seedlings were prepared by using Honda buffer supplement with RNase inhibitor and yeast tRNA to protect the integrity of RNA. The nuclei pellet was first resuspended in an equal volume of resuspension buffer (50% Glycerol, 0.5 mM EDTA, 1 mM DTT, 25 mM Tris-HCL pH 7.5, 100 mM NaCl, 100ng/ul tRNA, 0.01U/ul RNase In), followed by brief washing with two volumes of UREA wash buffer (25 mM Tris-HCL pH 7.5, 300 mM NaCl, 1 M urea, 0.5 mM EDTA, 1 mM DTT, 1% Tween-20). The chromatin was pelleted by centrifuge at 8000g for 1mins, resuspension with equal volume of resuspension buffer, followed by washing by using 1 volume of UREA buffer. The resulting chromatin pellet after centrifugation was resuspended in 1mL Trizol and followed by RNA extraction in combination with RNeasy Mini Kit (Qiagen).  The integrity of resulting CB-RNA was checked on gel by comparing to the pattern of total RNA.  DNA leftover was removed from CB-RNA by two sequential digestions with Turbo DNase (Thermo).  The resulting RNA were then used for further analysis of the enrichment of nascent RNA (qPCR) and constructing the RNA-seq library. iCLIP-seq : Plant growing on petri-dish for 10 days were immersed in an sallow layer of cold 1xPBS and cross-link with UV light at 600mJ/cm once. 2g of N2 grinded plant powers were lysed with lysis buffer, DNase RNase treated and sonicated to release the RNA/proteins. The resulting lysate were cleared with centrifugation, subject to immunoprecipitation with anti-GFP antibody (Ab290, Abcam) and protein A (Dynabeads, Thermo). CB-RNA-seq library: CB-RNA was treated with Ribozero (Illumina) to remove the rRNA following the kit instruction. Leftover polyA RNAs were also removed by using Oligo d(T) cellulose beads (NEB). Strand specific RNA-seq library were then constructed by using dUTP method. iCLIP-seq library: The RNA/protein complex after IP were washed extensively including several wash with high salt wash buffer, treated with PNK and Alkinine phosphatase to remove the terminal phosphate, ligated to RNA adaptor at 3'end and end labeled with p32.  90% of the resulting complex were eluted by LDS sample buffer and resolved on 4-12% NuPAGE gel, transfer to nitrocellulose membrane for autoradiograph.  The rest 10% of the complex were run in parallel to check the enrichment of target protein.  The region above the size of proteins were cutted out based on the autoradiograph,  RNA were released by proteinase K digestion.  The resulting RNA was further purified and reverse transcribed by Superscript III (Thermo) using primer complement to the RNA adaptor.  The resulting cDNA were purified and subject to adaptor ligation to its 3'end.  The final library was amplified by using primers specific to both end adaptors from cDNA by Q5 hotstart DNA polymerase. For Input sample, the supernatant after cell lysis were treated the same as iCLIP procedure as described above except the IP were omitted. The resulting PCR product were resolved on 6% native PAGE gel and fragment from 175-400bp were recovered from gel and send for pair-end illumina (PE150) sequencing.
identifiers.namespace: GEO
identifiers.id: GSM3681158