AGO CLIP reveals an activated network for acute regulation of brain glutamate homeostasis in ischemic stroke [dataset 7]

In vivo microRNA-target interactions from adult mouse cortex were identified with CLEAR-CLIP, a modified AGO HITS-CLIP approach that allows direct ligation of microRNA and target within purified, endogenous AGO-RNA complexes. Native AGO-RNA complexes were purified under stringent conditions selectively preserve cross-linked AGO-target interactions, then microRNA and target mRNA were ligated within the AGO complex to form miRNA-target chimeras in addition to standard AGO CLIP reads. AGO-bound RNAs were cloned by addition of a pre-adenylated 3' linker, a 5' RNA linker containing a 4 nt degenerate barcode followed by a G (NNNNG), and RT-PCR amplification. A second round of PCR amplification added a 4 nt index (for multiplexing) followed by a 16 nt common priming sequence. Raw data files have been de-multiplexed, but are otherwise not modified. DATA PROCESSING: base calling: Casava 1.8.2 quality filtering: minimum quality filter applied for barcode and insert; fastq_filter.pl; -if sanger -f min:0-3:20,mean:20-40:20 -maxN -1 -v -of fasta ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download collapse exact sequences: exact duplicate sequences removed; fasta2collapse.pl; ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download remove 3' adapter sequene: 3' adapter GTGTCAGTCACTTCCAGCGG clipped; fastx Clipper; -l 20 -a GTGTCAGTCACTTCCAGCGG -n; http://hannonlab.cshl.edu/fastx_toolkit/ trim 5' adapter sequence: 5' 20 nucleotides trimmed (NNNNAGGGAGGACGATGCGG, where NNNN is 4 nucleotide index for multiplexing); fastx Trimmer; -f 21; http://hannonlab.cshl.edu/fastx_toolkit/ strip 5' degenerate linker: 5 nucleotide (NNNNG) degenerate barcode stripped and appended to read IDs; stripBarcode.pl; -linker 5 ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download alignment against micoRNA database: microRNA database mapped against sample file (treated as reference genome) to identify potential chimeric reads containing microRNA sequence; Bowtie; -n 1 -l 8 -e 35; http://bowtie-bio.sourceforge.net/index.shtml extract chimeric read: extract potential chimeric sequence 5' or 3' of mapped microRNA sequence; text manipulation in R; ; alignment against genome reference: alignment ofpotential chimeric reads against reference genome to identify target sites; Bowtie; -n 1 -l 8 -e 35; http://bowtie-bio.sourceforge.net/index.shtml collapse PCR duplicates: mapped reads with same 5' end and same degenerate barcode consolidated; tag2collapse.pl; keep-tag-name --keep-max-score --random-linker -EM 30 --seq-error-model em-local --weight --weight-in-name ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download clustering : chimera interactions with the same ligated microRNA and overlapping genomic coordinates were clustered; Genome_Intervals R package; ; mm10 genomic coordinates of 126,295 CLEAR CLIP chimeras, chromosome, start, end, associated mmu miRNA, strand info