Altered activity-dependent nascent transcription in Rai1-knockdown mature mouse cortical cultures

We performed the nascent RNA-seq technique, Bru-Seq, and chromatin immunoprecipitation sequencing (ChIP-seq) for Retinoic acid induced 1 (RAI1) using a custom antibody, on mouse neuronal cultures treated with control or Rai1-targeted shRNA, with 4 hour treatment of vehicle, tetrodoxin (TTX) or bicuculline (BIC) and 20 minute labeling of bromouridine. 2-3 biological replicates, each consisting of cortices and hippocampi pooled from sex-ratio matched groups of E18 mouse embryos. Cells were plated and cultured for 14 days in Neurobasal + B27 medium, at which point lentiviral delivery of control or Rai1-targeted shRNA was performed. 3 days later, cells were treated with TTX, Bicuculline, or vehicle (water) for 4 hours. For Bru-seq: In the last 20 minutes, bromouridine was applied to the cells to label nascent RNA. Follow RNA purification, bromouridine-immunoprecipitation was performed to isolate nascent RNAs, preceding cDNA library synthesis. Pooled stranded libraries were run on Illumina Hiseq, single-end, 50 bp reads. For RAI1 ChIP-seq: Cells were harvested, fixed, and RAI1-bound chromatin isolated. Pooled libraries were run on Illumina NextSeq, single-end, 75 bp reads.