GSM8677104: 3tag_mRNAseq cdca7_ab_CDCA7_no_YFP, R1; Arabidopsis thaliana; RNA-Seq
Total RNA was extracted using the RNA isolation kit provided by the VBC core facilities. The kit uses a lysis step based on guanidine thiocyanate (adapted from (Boom et al., 1990) and carboxylate-modified Sera-Mag Speed beads and was applied using the KingFisher instrument (Thermo). For transcriptomes of cdca7 and ddm1 cdca7 mutants (Fig. 2, 3), we pooled three 31-day-old plants (two rosette leaves each), and used two or three biological replicates per genotype. For transcriptomes of complemented cdca7 mutants (Fig. 4, 5), we used one rosette leaf from 28-day-old plants. Libraries were prepared in 96-well plates using a custom-protocol described previously (Clavel et al., 2024). The oligo d(T) primers used for reverse transcription contain 8-bp unique molecular identifiers (UMIs), 7-bp barcodes for multiplexing, and a Truseq adapter. After reverse transcription, samples are pooled and RNA-cDNA duplexes tagmented with Tn5. Using primers matching the Truseq and Tn5 adapters, the 3' ends of mRNA transcripts are subsequently amplified by PCR, while P5 and P7 sequences and plate-specific dual indices are added for Illumina sequencing. This protocol improves speed and cost-efficiency compared with conventional library preparation protocols, and UMIs ensure accurate transcript quantification. We started from 80 ng of total RNA in 4 µl, adding 1 µl of 0.2 µM primers for reverse transcription. RNAs were denatured 3 min at 72°C and kept on ice. We mixed 1 µl of the SMARTScribe reverse transcriptase (Takara) in a final volume of 10 µl with 1x SMARTScribe buffer, 1.75 mM dNTPs, 2 mM DTT, 3 mM MnCl2 for 1h at 42°C followed by inactivation for 15 min at 70°C. All samples were pooled together and purified using 1.2x volume of SPRI beads (Molecular Biology Services, IMP, Vienna, Austria). After mixing and 2 min at room temperature, beads were collected on a magnet, washed two times with 70% ethanol, dried, and eluted in 60 µl of 10 mM Tris-HCl pH 8.0. To assemble transposomes, we mixed Tn5 (0.38 mg / ml) with 18 µM of annealed adapters, 50% glycerol and 1x Tn5 loading buffer (6x buffer: 300 mM Hepes KOH pH 7.2, 600 mM NaCl, 0.5 mM EDTA, 6 mM DTT, 0.6% Triton-X-100) and incubated for 1h at 37°C. For tagmentation, 16 µl of pooled RNA-cDNA was mixed with 16 µl of transposome and 32 µl of 2x tagmentation buffer (20 mM Tris (hydroxymethyl)aminomethane, 10mM MgCl2, adjusted to pH 7.6 and 20% (vol/vol) dimethylformamide). After 8 min at 55°C, we added 16 µl of 0.2% SDS and incubated 5 min at room temperature. Nucleic acids were purified with 2x SPRI bead volume as indicated above with a 48 µl elution. For PCR, we pre-incubated at 95°C the KAPA HiFi HotStart 2X ReadyMix for 90 s to activate the polymerase and amplified libraries in 100 µl final volume using 1 µM of primers and 46 µl of purified cDNA-RNA duplexes with the following program: 72°C for 3 min, 95°C for 30 sec, (95°C for 20 sec, 65°C for 30 sec, 72°C for 30 sec) for 12 cycles, 72°C for 5 min. Libraries were purified with 0.8x volume of SPRI beads as indicated above and eluted in 62 µl. After controlling for appropriate fragment size distribution on a 5200 Fragment Analyzer System (Agilent), libraries were sequenced as paired-end 150 bp reads on a NovaSeq S4 (Fig. 2, 3) or a NovaseqX 10B (Fig. 4, 5) flow cell.