GSM4188993: gastrula_GroSeq; Caenorhabditis elegans; OTHER
Staged embryos designated for Gro-seq analysis were prepared as described previously in Kruesi et al. 2013, with minor alterations.  First, we did not fragment nascent BrUTP-labeled RNA transcripts with sodium hydroxide, electing instead to keep full-length, nascent RNA molecules intact.  Second, we used the Ribo-Zero Gold rRNA Removal Kit (Illumina) to deplete rRNA transcripts from immunoprecipitated BrUTP-labeled nascent RNA in an effort to minimize noise and increase transcriptome coverage in our Gro-seq libraries Sequencing libraries were prepared from BrUTP-labeled RNA using the PCR-cDNA sequencing kit from Oxford Nanopore Technologies (cat: SQK- PCS109).  Libraries were prepared using ~100 ng of input RNA according to the manufacturer's specifications.  Briefly, full-length nascent RNAs are polyadenylated in vitro to initiate annealing of 3' poly-T oligo and reverse transcription by SuperScript IV Reverse Transcriptase (cat: 118090010).  Double-stranded cDNAs were size-selected to a range of 300-1000 bp using SPRI beads (AmpureXP) and were amplified with low-cycle PCR prior to sequencing.  Following limited PCR amplification, cDNA libraries were primed with rapid sequencing adapters and loaded onto the MinION flow cell.  We sequenced independent, replicate cDNA libraries from gastrula and late stage embryos—we later merged reads from gastrula and late stage replicate libraries into single fastq files, representing ~15 million total reads for each developmental stage Gro-Seq