GSM1821501
GSM1821501: RNASeq Vascular [TGACCA_V]; Sorghum bicolor; RNA-Seq
For all experiments carried out in this manuscript, Sorghum bicolor (L.) Moench (BTx623) seeds were separated into three biological replicates prior to germination under sterile conditions at ( 28C for N/NV LCM) while maintained in a 16/8 light/dark cycle for 3 days. For whole root and whole shoot transcriptomes RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer's instructions followed by DNase treatment using RQ1 enzyme (Promega). Whole root and whole shoot methylomes were prepared from DNA extracted using a method modified from that of Shure. Briefly, powdered tissue that had been ground in N2 was resuspended in an extraction buffer containing 8 M urea, 350 mM NaCl, 50 mM Tris-HCI (pH 7.5), 20 mM EDTA and 2% sarcosyl prior to  phenol chloroform extraction and precipitation with isopropanol. DNA was collected by centrifugation and cleaned following resuspension in TE with ammonium acetate/isopropanol precipitation. Precipitated DNA was collected by centrifugation rinsed with 75% ethanol and resuspended in TE. For root vascular and non-vascular nucleic acid preps Sorghum tissue was prepared for laser capture microdissection (LCM) as described. Shoots and roots were separated prior to fixation in EEA. Samples were dehydrated through an ethanol series prior to embedding in paraplast X-tra. 8-10 micron sections were mounted on PEN membrane slides (Leica) and vascular and nonvascular tissue were separated using the LCM setup described. For RNA extraction a RNAqueous-Micro Kit (Ambion) was used and for DNA extraction a QIAamp Micro DNA kit (Qiagen). For whole root and whole shoot RNA-seq, mRNA was purified from 1µg of total RNA using magnetic beads containing poly-T oligos. LCM RNA-seq libraries were prepared by removing rRNA from 10-50ng of total RNA using Ribo-Zero™ rRNA Removal Kit (Epicentre). For both whole shoot/root and LCM captured material, stranded cDNA libraries were generated using Illumina Truseq Stranded RNA LT kit (Illumina). mRNA was fragmented using divalent cations and high temperature. The fragmented RNA was reversed transcribed using random hexamers and SSII (Invitrogen) followed by second strand synthesis.  The fragmented cDNA was treated with end-repair, A-tailing, adapter ligation, and 10 cycles of PCR (for whole root/shoot) or 15 cycles of PCR (for LCM captured material).  qPCR was used to determine the concentration of the libraries. Libraries were sequenced on the Illumina Hiseq. Whole root and whole shoot methylomes were generated from 1ug of DNA which was sheared to 500bp using the covaris LE220 (Covaris).  DNA fragments smaller than 200bp were removed using SPRI beads (Beckman Coulter).  The fragments were treated with end-repair, A- tailing, and ligation of methylated Illumina adapters (Illumina) using an Illumina library creation kit (KAPA biosystems).  The adaptor ligated DNA were bisulfite treated using EZ DNA Methylation Lightning Kit (Zymo Research), converting the non-methylated nucleotides from Cytosines to Uracil. The converted DNA was enriched with 10 cycles of PCR to generate the final library. qPCR was used to determine the concentration of the libraries. Libraries were sequenced on the Illumina Hiseq. Vascular and non-vascular root methalomes were generated in the same manner, but  from 100ng of Laser Capture Microdissection DNA which was sheared to 700bp. Removal of < 200 bp fragments, end-repair, A- tailing, adaptor ligation and bisulphide conversion were as described for whole root/shoot. The converted DNA was enriched with 12 cycles of PCR to generate the final library.
GEO
GSM1821501
GEO Accession
GSM1821501