GSM9003564: Petunia axillaris, young bud, 9 weeks; Petunia axillaris; ncRNA-Seq
Total RNA was isolated by a standard method using TRIzol® Reagent (Life Technologies), including a second precipitation step to increase RNA purity, and using 20 minutes centrifugation, 14.000 rpm, to optimize small RNA recovery. Samples were stored in the second precipitation, in 0.3 M Sodium Acetate (NaAc)/70 % Ethanol (EtOH), at -80 °C, upon library preparation. RNA was suspended in 10-30 μl DEPC-MQ to a final concentration of ~ 1 μg/μl, and checked on agarose for a 25S to 18S ribosomal peak ratio of > 4:5 to be used for library preparation. Small RNA libraries were prepared from 1.2 μg total RNA, using the Illumina compatible NextFlex™ Small RNA Sequencing Kit (version 1; Bioo Scientific; Sanbio BV, Uden, The Netherlands; Cat. no. 5132) and accompanying Small RNA Barcodes Primers (Cat. no. 5133). This protocol included an 18 h long 3'-adapter ligation of pre-adenylated oligonucleotides, with a modified ligase, to optimize binding to plant miRNAs and other small RNAs that contain 2'-O-methylated 3'-ends (Munafó and Robb, 2010; McCormick et al., 2011). After the 5'-adapter ligation, the recommended purification step was included, using the RNA Clean and Concentrator Kit (Zymo Research; BaseClear, Leiden, The Netherlands; Cat. no. R1015). We amplified the cDNAs by 14-16 PCR cycli. The ~ 150 nt long small RNA libraries were isolated from 3 % NuSieveTM (Lonza, Basel, Switzerland)/1 % agarose gels, and extracted by the QIAquick Gel Extraction Kit (Qiagen Benelux BV, Venlo, The Netherlands; Cat. no. 28706). Libraries were eluted in 40μl pre-warmed buffer of the Kit, and precipitated in NaAc/EtOH, at -20°C, after addition of 1 μl co-precipitant of the NextFlex Kit. After re-suspension of the small RNA libraries in 10 μl MQ, they were sent to BaseClear (Leiden, The Netherlands) for Illumina HiSeq 2500 sequencing. Quality Control included a BioAnalyzer measurement (Agilent Technologies) and qPCR, on which basis the samples were pooled in equal amounts to 18 samples/lane in order to provide a minimum of ~ 5 Million Reads/library. Sequence data was de-multiplexed and reads delivered as '.fastq' files together with the run-statistics.
GEO
GSM9003564_r1