title: GSM3330789: Ground control, elp2-5, Roots, replicate 4; Arabidopsis thaliana; Bisulfite-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. DNA extraction was done using a modified phenol/chloroform protocol (LeFrois et al., 2016) and genomic-wide bisulfite sequencing was performed using a similar procedure as described by (Wang et al., 2013). 700-1700 ng of genomic DNA (>5 Kb in length) observed on the TapeStation Genomic Screen Tape (Agilent) was processed for sequencing library construction. Library preparation:  DNA was transferred into 6x16 mm glass microtubes with AFA fiber and pre-slit snap caps (Cat# 520045, Covaris, Inc.) and sheared into average fragments size of ~400 bp, using the Covaris S220 ultrasonic disruptor. Short DNA fragments (<100 bp) were removed using AMPure magnetic beads (Cat# A63881, Beckman Coulter) at a 1:1 bead to sample ratio.  100-250 ng of clean, fragmented DNA was used for the Illumina sequencing library construction.  The NEBNext® UltraTM II DNA Illumina construction kit (Cat# E7645S, NEB) in conjunction with the Illumina-specific methylated and dual-index barcoded adaptors (Cat# E7600S NEB) were used as described in the manufacturer's guidelines. Illumina libraries (containing methylated adaptors) were subjected to sodium bisulfite treatment using the EZ DNA Methylation Direct kit (ZYMO Research, Cat #D5020) according to the manufacturer's instructions. The resulting libraries were enriched by a 13-15 cycle amplification using an uracil-insensitive polymerase (EpiMark hot start Taq polymerase, NEB, Cat#M0490S). The amplified library products were separated on a 2% agarose gel from which library fragments in the 250-500 bp range were excised (QIAquick gel extraction kit, Cat# 28704, QIAGEN) and AMPure purified (Cat# A63881, Beckman Coulter). Gel staining was done with SYBR safe (Life Technologies) and visualized on a blue light transilluminator (Life Technologies) to avoid UV damage to the DNA. The final libraries were quantitated by the QUBIT fluorometer, sized on the Agilent TapeStation (DNA5000 Screen Tape) and by qPCR with the Kapa SYBR Fast qPCR reagents (Cat# KK4824, Kapa Biosystems) with monitoring on an ABI7900HT real-time PCR system (LifeTechnologies). The average library size was 350 bp. Care was taken to generate WGBS libraries that were approximately the same size as the RNA-Seq libraries. 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.