title: GSM4704327: undifferentiated PAT-Seq2; Homo sapiens; OTHER
library_construction_protocol: Poly(A) tail sequencing (PAT-Seq) was conducted using 1µg of high quality RNA (RNA integrity number = 10 for all samples) as previously described (Harrison et al, 2015). Briefly, biotinylated ePAT primers (5´-Bio-CAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTT-3´; 100µM) were ligated to polyadenylated mRNAs and 3´ ends were extended using 5 units (U) of the Klenow fragment of DNA polymerase I (NEB, Ipswich, MA, USA). RNAs were then subjected to limited RNase T1 digestion (10U, Roche, Basel, Switzerland), with parameters optimised to produce ~250nt fragments, after which 3´ fragments were collected with streptavidin beads (NEB). Phosphorylation of 5´-ends was next performed with Poly Nucleotide Kinase (0.5U, NEB), prior to ligation of 5´ splinted linkers, reverse transcription and size selection (120–400bp) via 6% denaturing urea-polyacrylamide gel. To amplify all samples, 10µL of cDNA was subjected to 16 PCR cycles, with 2µL of ScriptSeq indexed reverse primers (50µM, Epicentre, Madison, WI, USA) included for sample indexing compatible with Illumina (San Diego, CA, USA) sequencing platforms. PAT-Seq libraries were subsequently normalised, pooled and sequenced on a single lane of an Illumina HiSeq 1500, with a total of 151 single ended cycles performed. Total RNA was extracted from all groups by harvesting cells in 1mL TRIzol reagent (ThermoFisher, Waltham, MA, USA), as per the manufacturer's instructions, with RNA precipitated at –30°C overnight to increase yield.
attributes.tag: GEO Accession
attributes.value: GSM4704327