title: GSM1678912: S2_GROseq_r2; Drosophila melanogaster; OTHER
library_construction_protocol: 5’GRO-seq was performed as described previously (Lam et al., 2013). About 10^7 Drosophila S2 nuclei were used for run-on with BrU-labeled NTPs. Fragmented transcripts were incubated with polynucleotide kinase (PNK, NEB) to remove 3’ phosphates. BrU-labeled nascent transcripts were subsequently immunoprecipitated with anti-BrdU agarose beads (Santa Cruz Biotech). For 5’GRO-seq, immunoprecipitated RNA was dephosphorylated with calf intestinal phosphatase (NEB). Then 5’ capped fragments were de-capped with tobacco acid pyrophosphatase (Epicenter). Illumina TruSeq adapters were ligated to the RNA 3’ and 5’ ends with truncated mutant RNA ligase 2 (K227Q) and RNA ligase 1 (NEB), respectively. Reverse transcription was performed with Superscript III (Invitrogen) followed by PCR amplification for 12 cycles. Final libraries were size selected on PAGE/TBE gels to 175–225 bp. GRO-seq was essentially performed as 5’GRO-seq, but the immunoprecipitated RNA was directly de-capped with tobacco acid pyrophosphatase (Epicenter) and subsequently kinased with PNK (NEB) prior to adaptor ligation. 5’GRO-seq was performed as described previously (Lam et al., 2013). About 10^& Drosophila S2 nuclei were used for run-on with BrU-labeled NTPs. Fragmented transcripts were incubated with polynucleotide kinase (PNK, NEB) to remove 3’ phosphates. BrU-labeled nascent transcripts were subsequently immunoprecipitated with anti-BrdU agarose beads (Santa Cruz Biotech). For 5’GRO-seq, immunoprecipitated RNA was dephosphorylated with calf intestinal phosphatase (NEB). Then 5’ capped fragments were de-capped with tobacco acid pyrophosphatase (Epicenter). Illumina TruSeq adapters were ligated to the RNA 3’ and 5’ ends with truncated mutant RNA ligase 2 (K227Q) and RNA ligase 1 (NEB), respectively. Reverse transcription was performed with Superscript III (Invitrogen) followed by PCR amplification for 12 cycles. Final libraries were size selected on PAGE/TBE gels to 175–225 bp. GRO-seq was essentially performed as 5’GRO-seq, but the immunoprecipitated RNA was directly de-capped with tobacco acid pyrophosphatase (Epicenter) and subsequently kinased with PNK (NEB) prior to adaptor ligation. Illumina HiSeq 2500 system and associated protocol was used. Run type: SE50; Run mode: High Output
attributes.tag: GEO Accession
attributes.value: GSM1678912