GSM6604867: Quiescent 1, Roots, KFT A, Replicate 3; Arabidopsis thaliana; RNA-Seq
RNA extraction was performed using Qiagen RNeasy Plant mini kit (Qiagen, Hilden, Germany catalog # 74904) according to the manufacturer's guidelines. RNA concentration was determined on Qubit 2.0 Fluorometer and RNA quality was assessed using the Agilent 2100 Bioanalyzer (AgiRNA extraction was performed using Qiagen RNeasy Plant mini kit (Qiagen, Hilden, Germany catalog # 74904) according to the manufacturer's guidelines. RNA concentration was determined on Qubit 2.0 Fluorometer and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). First, 100 ng of total RNA was used for mRNA isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog # E7490). Then followed by RNA library construction with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, catalog #E7760) with 2/3 of poly A enriched RNA and 1/10 reaction volume using SPT Labtech mosquito LV liquid handling instrument. Briefly, RNA was fragmented in NEBNext First Strand Synthesis Buffer via incubation at 94 °C for the desired time. This step was followed by first-strand cDNA synthesis using reverse transcriptase and random hexamer primer.  Synthesis of ds-cDNA was performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. At this point, Illumina adaptors were ligated to the sample.  Finally, each library (uniquely barcoded) was enriched by 15 cycles of amplification, and purified with Agencourt AMPure beads (Beckman Coulter, catalog # A63881). Barcoded libraries were sized on the Bioanalyzer and quantified with the Qubit 2.0 Fluorometer. Finally, these individual libraries were pooled in equimolar concentration for sequening aiming for 60 million reads per sample with total of 1 lane of NovaSeq S4 2x150 cycles run. RNASeq libraries were constructed at the UF ICBR Gene Expression Core (https://biotech.ufl.edu/gene-expression-genotyping/, RRID:SCR_019145). The Illumina NovaSeq 6000 was used to sequence the libraries for 2 x 150 cycles. Sequencing was performed at the ICBR NextGen Sequencing (https://biotech.ufl.edu/next-gen-dna/, RRID:SCR_019152). Sequencing was performed at the ICBR NextGen Sequencing Core (https://biotech.ufl.edu/next-gen-dna/, RRID:SCR_019152). Normalized libraries were submitted to the “Free Adapter Blocking Reagent” protocol (FAB, Cat# 20024145) in order to minimize the presence of adaptor-dimers and index hopping rates.  The library pool was diluted to 0.8 nM and sequenced on one S4 flow cell lane (2x150 cycles) of the Illumina NovaSeq6000. The instrument's computer utilized the NovaSeq Control Software v1.6. Cluster and SBS consumables were v1.5. The final loading concentration of the library was 120 pM with 1% PhiX spike-in control. One lane generated 2.5-3 billion paired-end reads (~950Gb) with an average Q30%>= 92.5% and Cluster PF= 85.4%. FastQ files were generated using the BCL2fastQ function in the Illumina BaseSpace portal.