title: GSM4761303: N2_COPAS_earlyL4_rep1_GRO-seq; Caenorhabditis elegans; OTHER
library_construction_protocol: Synchronous populations of L1 worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish. Worms were collected at the indicated developmental stage: early L4 ( 38 hours post hatching (hph)), late L4 (44hph) or Young Adult ( 48hph). The harvested worms were washed three times with M9 buffer and nuclei were extracted in Nuclei extraction buffer using a metal dounce on ice. GRO-seq experiments were performed as described by Barucci et al., 2014 with minor modifications. Biotinylated nascent RNAs were fragmented and immunoprecipitated using streptavidin conjugated beads (Invitrogen, 65001). 5'-OH of fragmented RNAs was repaired using Polynucleotide kinase (New England biolabs, M0201S). RNA was ligated to 3'end adapter using T4 RNA ligase 2 Truncated KQ and subjected to a second round of immunoprecipitation with streptavidin beads. RNA was ligated at 5' end using T4 RNA ligase 1 (New England biolabs, M0204S) and biotinylated RNA was enriched for a third time. Purified RNA was reverse transcribed using SuperScript IV Reverse Transcriptase (Invitrogen, 18091050). cDNA was PCR amplified with specific primers using Q5 High-Fidelity 2X Master Mix (New England biolabs, M0492S). The purified libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced either on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).