The detailed methods of CHIRP assay were previously described [12 (link)]. Briefly, sonicated chromatin materials diluted using hybridization buffer and hybridized with 100pmol of biotinylated tiling DNA probes targeting HOTTIP or LacZ containing 100 μL of Streptavidin-magnetic C1 beads (Invitrogen). RNA and DNA hybrids and RNA binding proteins were subjected to analysis by ChIRP-qPCR and ChIRP-RT-qPCR. Illumina’s TruSeq ChIP Sample Preparation Kit was used according to manufacturer’s instructions for preparation of CHIRP libraries and submitted to paired-end sequencing of 100 bp length on an Illumina HiSeq 2500. All sequencing genomics datasets were deposited in the NCBI GEO under accession number (GSE114981).
Protocol detail
CHIRP Assay for lncRNA Binding Analysis
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Assay
Bp 100
Buffer
Chip
Chromatin
Dna probes
Hybridization
Hybrids
Lacz
Rna binding proteins
Streptavidin
Corresponding Organization : Pennsylvania State University
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Variable analysis
independent variables
- Biotinylated tiling DNA probes targeting HOTTIP or LacZ
- RNA and DNA hybrids
- RNA binding proteins
- Streptavidin-magnetic C1 beads (Invitrogen)
- Hybridization buffer
- Sonicated chromatin materials
- HOTTIP
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