Zea mays B73 seedling primary and seminal root tips (0-1mm) RNA-seq, Nuclear and EU-nascent transcription data
Zea mays B73 seedlings were grown for 3 days at 28 C under continuous dim light (~500 lux). Three day old seedlings were conditioned for 1 hour in water at 28 C and then labeled for 1 hour at 28 C in 175 uM EU. Terminal 0-1 mm root segments from both the primary and seminal roots were excised on ice and fixed in 1% formaldehyde. Nuclei were isolated by chopping the root tips with a single edged razor blade. RNA was isolated from the fixed nuclei using a Qiagen RNeasy FFPE Kit. DNA was removed using an Invitrogen Turbo DNA Free Kit. Ribosomal RNA was depleted using a Vazyme Ribo-Off rRNA depletion kit for plants. The RNAs containing EU were biotinylated using an Invitrogen Click-it Nascent RNA capture kit. One tenth of the RNA sample was kept back as a nuclear steady state input control while the remaining RNA was incubated with streptavidin beads using the Invitrogen Click-it Nascent RNA capture kit to enrich for biotinylated EU-RNAs. First strand cDNA synthesis was performed on both the nuclear RNA control and the nascent EU-RNA samples using a Superscript IV First Strand Synthesis System using random hexamer priming. RNA-seq libraries were made using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (starting at step 1.4.1 because cDNAs were previously made) in combination with NEBNext Multiplex Oligos for Illumina, Unique Dual Index UMI Adaptors RNA Set 1. The protocol to target 200 nt inserts was followed and the adapter ligated libraries were amplified for 14 PCR cycles. The average library size (insert + 150 bp adapter) ranged from 390-424 bp making the average insert size 240-274 bp. Libraries were sequenced using 150 bp PE on one lane of the NovaSeq 6000 S4 flow cell, using custom i7 cycles of 19 cycles to pick up the i7 index (8 nt) plus the 11 nt UMI. The UMI sequences have been appended to the read headers in the R1 and R2 files during demux with BCL Convert and after read alignment can be used by software, such as UMItools, to identify PCR duplicate reads.