GSM8343190: set2.urt1_rep3; Arabidopsis thaliana; RNA-Seq
Inflorescences and rosettes for northern blots, western blots and sequencing libraries were collected and snap-frozen in liquid nitrogen. Total RNA from inflorescences was extracted with TRI Reagent (#T9424, Sigma-Aldrich Denmark A/S) according to the manufacturer's instructions. The RNA was once again resuspended and purified with phenol:chloroform:isoamyl alcohol. The RNA was dissolved in nuclease-free water and RNA integrity was assessed on a Bioanalyser RNA Nano Chip (Agilent Technologies). 10 μg of total inflorescence RNA was used as input in each library. Twenty pmol of a 3'-ssDNA adapter (custom order, IDT), previously described in (Zuber et al., 2018) was ligated to 10 μg of total RNA. To separate excess adapter from successfully ligated RNA, samples were purified using NucleoSpin® RNA Clean-up columns (Macherey Nagel). Next, 2 x 20 μl reactions of 2 μg of denatured RNA incubated with 50 pmole of 3'-RT oligo, 10 mM dNTP mix, 200 U of SuperScript IV reverse transcriptase (InvitrogenTM), 1X SuperScript RT buffer, 0.1 M DTT and 40 U Ribolock (ThermoScientific) were incubated at 50°C for 10 minutes. The reverse transcriptase was then inactivated at 80°C for 10 min. The cDNA was purified with phenol:chloroform:isoamyl alcohol and the aqueous phase was EtOH-precipitated. The cDNA was resuspended in 8μl of water. Two rounds of nested PCR reactions were then performed. The first PCR contained 2 μl of cDNA, 10 pmol of a gene-specific primer (MYB33_5'CF/AFB2_5'CF/AFB2_3'CF), 10 pmol of RACEseq_rev1 primer, 12.5 nmol dNTP mix, 1U of Phusion DNA Polymerase and 1 x Phusion HF buffer (ThermoScientific). The PCR specifications were 94 °C 30 sec, (94°C 20 sec, 50°C 20 sec, 72°C 30 sec) x 20 cycles, 72 °C 30 sec. Thereafter, 4 x 20 μl reactions for each gene-specific PCR reaction were set up using 1 μl of PCR reaction 1, 10 pmole of a new gene-specific primer, 10 pmole of TRUseq RPI primer, 12.5 nmole of dNTP mix, 1 U Phusion DNA Polymerase and 1 x Phusion HF buffer (ThermoScientific). The PCR specifications were 94°C 1 min, (94°C 30 sec, 56°C 20 sec, 72°C 30 sec) x 25 cycles, 72 °C 30 sec. The four identical PCR reactions were pooled, and the libraries were PCR purified using Beckman CoulterTM Agencourt AMPure XP beads following the manual from the provider. The libraries were assessed with Agilent Bioanalyser on a DNA 1000 chip and the concentration was determined with Qubit HS DNA chemistry (InvitrogenTM). Libraries were pooled in equimolar ratios and sequenced on a NextSeq (v3 chemistry) MidOutput flow cell with 41 x 111 bp settings. 50% of phiX was added to compensate for the poor read diversity of poly(A)-tails. Sequences of all adapters and primers used for the library construction are listed in Dataset EV3 in https://doi.org/10.1101/2024.05.27.596008.