title: GSM4576979: S_47.1: myb36-2_SynCom_BioRep1_S47TechRep2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: RNA was extracted from A. thaliana roots according to Logemann et al. We used three independent biological replicas per treatment and we repeated this experiment twice. Briefly, approximately 10 roots from 14-day-old seedlings were harvested from each sample and flash frozen using liquid N2. Frozen roots were pulverized using a TissueLyzer II (Qiagen), 2 cycles of 30 seconds, frequency 30 s-1, and 400 μL of Z6-buffer; 8 M guanidine HCl, 20 mM MES, 20 mM EDTA at pH 7.0 was added to the samples. Following the addition of 400 μL phenol:chloroform:isoamylalcohol; 25:24:1, samples were vortexed and centrifuged (20,000 g, 10 min) for phase separation. The aqueous phase was then transferred to a new 1.5 mL tube and 0.05 volumes of 1 N acetic acid and 0.7 volumes 96% ethanol were added. The RNA was precipitated at −20 °C overnight. Following centrifugation, (20,000 g, 10 min, 4 °C) the pellet was washed with 200 μL sodium-acetate (pH 5.2) and 70% ethanol. The RNA was dried and dissolved in 30 μL of ultrapure water and stored at −80 °C until use. Before sequencing, RNA samples were quantified using the Qubit RNA BR Assay Kit (Invitrogen; Q10210) and quality was assessed using RNA Screen Tape (Agilent; 5067-5576) on the Agilent 4200 TapeStation System (Agilent; G2991A). Although TapeStation results showed RNA with high RNA integrity numbers (RINs > 8), indicative of good quality RNA, we detected the presence of high molecular weight nucleic acids typical of genomic DNA, therefore DNase treatment was carried out on the samples. TURBO DNase (Ambion; AM2238) was used to remove DNA from RNA samples. Briefly, 22.5 µL of RNA was combined with 2.5 µL of 10x TURBO DNase Buffer and 1 µL (2 Units) of TURBO DNAse and incubated at 37°C for 30 min. The RNA was then dissolved with 75 µL of nuclease free water and purified using the Qiagen RNeasy MinElute Cleanup Kit (Qiagen; 74204). The final elution was carried out with 15 µL of nuclease free water. For library construction, 200 ng of high-quality RNA was used as input into Lexogen Quant Seq 3' mRNA Seq (FWD) Library Prep Kit (Lexogen; 015) and the standard protocol was followed. At the PCR stage 13 cycles of PCR were used. Library yield was measured by Qubit dsDNA HS (Invitrogen; Q32851) and library size was assessed using the High Sensitivity D1000 ScreenTape (Agilent; 5067- 5584) on the Agilent 4200 TapeStation System (Agilent; G2991A). Libraries were normalised to each other based on size and Qubit dsDNA HS concentration and pooled together. The pool was quantified by qPCR using the KAPA Library Quantification Kit for Illumina Platforms (KAPA Biosystems; KK4873) The library pool was denatured and diluted according to the Illumina NextSeq System protocol (Illumina Document #15048776v09) and then sequenced on the Illumina NextSeq 500 on a NextSeq 500/550 High Output Kit v2.5 (75 Cycles) (Illumina; 20024906), to generate ~5 million 75bp single-end reads per sample