GSM1848706: 36_BSseq_nrpb2_b; Arabidopsis thaliana; Bisulfite-Seq
Total RNA was first treated with RiboMinus™ Plant Kit for RNA-Seq (Invitrogen A10838-08) to remove rRNA, followed by size selection of RNA on a 15% UREA TBE Polyacrylamide gel (Invitrogen, EC6885BOX). Gels containing RNA with size between 15- to 27-nt were kept for small RNA library, while gels containing 28- to ~300-nt RNA were kept for PATH library. ChIP-seq was performed as described previously (Johnson et al., 2014). 5 ug Pol II antibodies (Abcam #ab817) were used for each ChIP. Libraries for Pol II ChIP-seq were generated using the Ovation Ultralow DR Multiplex System (NuGen #0330) and sequenced at a length of 50 bp. ChIP-seq data was visualized using ngsplot (Shen et al., 2014). AGO4-RIP was performed as previously described (Ji et al., 2011) with commercial AGO4 antibody (Agrisera #AS09 617). RNA isolated from RIP was used for library construction with Illumina TruSeq RNA Sample Preparation Kit (RS-122-2001) according to the standard manual. For BS-seq, DNA was isolated using the DNeasy Plant Mini Kit (Qiagen #69104) according to manufacturer instructions and quantified using the Qubit dsDNA High Sensitivity Kit (Life Technologies #Q32851). Libraries were constructed with 30ng DNA using the Ovation Ultralow Methyl-Seq Library Systems (NuGEN #0335). Bisulfite conversion was done using the EpiTect Bisulfite Kit (Qiagen # 59104). BS-seq Libraries were sequenced at a length of 50 bp. After gel elution, library construction for both sRNA and PATH was done using the Illumina TruSeq Small RNA Sample Preparation Kit (RS-200-0012), except that at the final size selection step, PCR products were separated on a 6% TBE Polyacrylamide gel (Invitrogen, EC6265BOX) and selected for the range from 120- to ~1000-bp for PATH library. Gel-eluted PCR products with different TruSeq index sequences were pooled and sent for Illumina sequencing. The mRNA library was constructed using the Illumina TruSeq RNA Sample Preparation Kit (RS-122-2001) according to the standard manual. PATH libraries were sequenced using either paired-end mode with length of read1 being 120-bp and length of read2 being 30-bp (PE120+30), or single-end 100-bp (SE100); while sRNA and mRNA libraries were sequenced with single-end 50-bp (SE50). All sequencing was carried out on HiSeq2500 machines at the Broad Stem Cell Research Center (BSCRC) sequencing core at University of California, Los Angeles.
GEO
GSM1848706