GSM2493768: Dark grown, Ground Control, Col-0 Rep1; Arabidopsis thaliana; RNA-Seq
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, root tip and then the rest of the root. The root tip was defined as the last 2mm of the root for the light-grown plants and the last 1mm for the dark-grown plants. All the sections dissected were saved in RNA later and stored back to -80°C freezer.  Three independent roots were used as three biological replicates for the transcriptomic analysis. The root apex was dissected as 2mm root tip section for light grown samples and 1mm section for the dark grown samples. Dissected root tips were carefully transferred into pre-labeled micro-centrifuge tubes, flash frozen in liquid N. Total RNA was extracted from these frozen samples using Picopure RNA isolation kit (Arcturus®, Life technologies). RNA concentration was determined on a Qubit (Thermo Fisher/Life Technologies, Inc) and sample quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Five ng RNA from each sample was used for low input array and RNA sequencing. RNASeq Libraries Preparation using SMART-Seq V4 ultra low input RNA kit for sequencing combined with Illumina Nextera DNA Sample Preparation Kit  Five ng of total RNA was used for cDNA library construction using CloneTech SMART-Seq V4 ultra low input RNA kit for sequencing (Clontech Laboratories, Inc, cat#: 634890) according to manufacturer's protocol. Briefly, 1st strand cDNA was primed by the SMART-Seq v4 oligonucleotide and then base-pairs with these additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide, resulting full-length cDNA that contains an anchor sequence that serves as a universal priming site for second strand synthesis. cDNA was amplified with primer II A for 9 PCR cycles. Then Illumina sequencing libraries were generated with 120 pg of cDNA using Illumina Nextera DNA Sample Preparation Kit (Cat#: FC-131-1024) according to manufacturer’s instructions. Briefly, 120 pg of cDNA was fragmented by tagementation reaction and then adapter sequences added onto template cDNA by PCR amplification. Libraries were quantitated by Bioanalyzer and qPCR (Kapa Biosystems, catalog number: KK4824). Finallly, the libraries were pooled equal molar concentration and sequenced by Illumina 2X75 NextSeq 500. NextSeq500 sequencing: RNA seq  In preparation for sequencing, barcoded libraries were sized on the bioanalyzer, quantitated by QUBIT and qPCR (Kapa Biosystems, catalog number: KK4824). Individual samples were pooled equimolarly at 4 nM. This “working pool” was used as input in the NextSeq500 instrument sample preparation protocol (Illumina, Part # 15048776, Rev A). Typically, a 1.3 pM library concentration resulted in optimum clustering density in our instrument (i.e., ~200,000 clusters per mm2). Samples were sequenced on three flowcells, using a 2x75 cycles (paired-end) configuration. A typical sequencing run in the NextSeq500 produced 750-800 million paired-end read with a Q30>=85%. For RNA seq, around 40 million reads provided sufficient depth for transcriptome analysis.