GSM2101782: siEGFP; Homo sapiens; OTHER
RNA was extracted with TRIzol (Invitrogen). Protocol based on Pelechano et al. Nat Protoc 2014. Briefly, 5’-to-3’ mapping of capped and poly(A) transcripts was carried out using the TIF-seq protocol from Pelechano et al. (PMID: 24967623). Briefly, total RNA was de-phosphorylated to remove any RNA degradation intermediates that may carry a 5’ phosphate, followed by a tobacco acid pyrophosphatase (TAP) treatment to remove the cap structure and expose the resulting 5’ phosphate for ligation of a single-stranded RNA oligo (used later for primer annealing). Each RNA sample was split in two independent tubes and subjected to reverse transcription followed by generation of double-stranded full-length cDNA by PCR. During this process a tube-specific 4nt sequence is introduced at each end of the cDNA fragments (chimera controls) that allow the identification of inter-molecular ligation events. During these process, we introduce a random octamer sequence at the 3’ end (unique molecular identifier, to control for PCR amplification) and a NotI site to facilitate the circularization and a biotin. Please see Pelechano et al. (PMID: 24967623) for a detailed description. The independently barcoded full-length cDNA samples were then pooled, digested (using NotI) to create sticky ends, and circularized overnight with T4 DNA ligase in conditions favouring intra-molecular ligation. Non-circularized molecules were degraded and circularized cDNAs fragmented by sonication. Biotin containing cDNA fragments were captured using streptavidin conjugated magnetic beads, and followed by standard Illumina library construction (including DNA blunt ending, dA addition, and ligation of forked adapters containing 6 nt barcodes identifying each sample) on beads. Barcoded libraries were pooled and sequenced paired-end (101 bp) at the EMBL Genomics Core Facility using an Illumina HiSeq 2000. Since our main interest was to identify transcript boundaries for PROMPT that are generally short compared with mRNA, no size selection to enrich for long RNA fragments was done.
GEO
GSM2101782