title: GSM3331118: met1-7, Roots, Ground control, replicate 4 [MRG4]; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Seedlings stored in RNA later were recovered from -80°C freezer. Each tube containing the seedlings was allowed to thaw in the fridge overnight. After completely thawing the samples to room temperature, the seedlings were observed under the microscope. The preserved seedlings from the spaceflight and ground control harvests were dissected into distinct plant parts: leaf, hypocotyl, and root. Total RNA extraction: Total RNA was extracted using Qiashredder and RNAeasy™ kits from QIAGEN (QIAGEN Sciences, MD, USA) according to the manufacturer's instructions. Residual DNA was removed by performing an on-column digestion using an RNase Free DNase (QIAGEN GmbH, Hilden, Germany). Integrity of the RNA was evaluated using the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). Library preparation:  RNAseq library were constructed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations. Basically, 2ul of of 1:200 diluted RNA spike-in ERCC (half amount of suggested in the ERCC user guide: Cat# 4456740) spike to 1000 ng of total RNA followed by mRNA isolating using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs, catalog # E7490). Then followed by RNA library construction with NEBNext Ultra Directional Lib Prep (New England Biolabs, catalog #E7420) according to the manufacturer's user guide. Briefly, RNA is fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C for desired time. This step is followed by first strand cDNA synthesis using reverse transcriptase and oligodT primers.  Synthesis of ds cDNA is performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation.  At this point, Illumina adaptors are ligated to the sample.  Finally, library is enriched (each library has a unique barcode) by 10 cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, catalog # A63881). 48 barcoded libraries were sized on the bioanalyzer, quantitated by QUBIT. Finally, 48 individual libraries were pooled with equimolar. Illumina sequencing:  Sequencing experiments were performed at the Interdisciplinary Center for Biotechnology Research (ICBR) gene expression and sequencing core, University of Florida. Uniquely barcoded libraries were normalized to 2.5 nM and pooled (equimolarly) for sequencing on the HiSeq3000 Illumina sequencer. Bisulfite-converted sequencing libraries were sequenced together with RNAseq libraries (uniquely barcoded) to maximize data output. The RNAseq libraries in the pool served to compensate for the low base diversity of bisulfite-converted genomic libraries. The final library was created by mixing RNA-seq vs bisulfite-converted libraries at a 60%:40% ratio, with a mere 1% PhiX spike-in. Library pools were processed according the Illumina protocol (HiSeq3000) for clustering on the cBOT machine. After denaturation, neutralization and mixing with the ExAmp reagent, the final pool concentration for clustering was 0.25 nM. Sequencing was done using a 2x101 cycles format (paired-end configuration). The 48-sample project was sequenced on 12 lanes for a robust reads/lane output.
attributes.tag: GEO Accession
attributes.value: GSM3331118