The ChIRP-seq assay was performed as described previously50 (link). Mouse ES cells were cultured as above and treated with Dimethyl sulfoxide (DMSO) or 500nM JQ148 (link) (Gift from J. Bradner) for 1 hour at 37°C. Isolated RNA was used in qRT-PCR analysis (Stratagene) to quantify enrichment of 7SK and depletion of other cellular RNAs. Isolated DNA was used for qPCR analysis or to make deep sequencing libraries with the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Library DNA sequenced from a single end for 75 cycles on an Illumina HiSeq 2500 (Summary of all sequencing experiments are listed in Supplementary Table 5 ).
Sequencing reads were first trimmed of adaptors (FASTX Toolkit) and mapped using Bowtie to a custom bowtie index containing repetitive RNA elements (rRNA, snRNAs, and y-RNAs51 ). Subsequent reads were then mapped to mm9. Mapped reads were separately shifted towards the 3′ end using MACS and normalized to a total of 10 million reads. Even and Odd replicates were merged as described previously50 (link) by taking the lower of the two read density values at each nucleotide across the entire genome. The full pipeline is available athttps://github.com/bdo311/chirpseq-analysis .
Sequencing reads were first trimmed of adaptors (FASTX Toolkit) and mapped using Bowtie to a custom bowtie index containing repetitive RNA elements (rRNA, snRNAs, and y-RNAs51 ). Subsequent reads were then mapped to mm9. Mapped reads were separately shifted towards the 3′ end using MACS and normalized to a total of 10 million reads. Even and Odd replicates were merged as described previously50 (link) by taking the lower of the two read density values at each nucleotide across the entire genome. The full pipeline is available at
