GSM5734421: glioma stem cells; Homo sapiens; RNA-Seq
To sequence full-length polyA and non-polyA LCT transcripts, a modified Iso-seq protocol (PacBio) was used. For each sample, total RNA was poly-uridinylated using a polyU polymerase (New England Biolabs) following the manufacturer's recommendations. PolyU RNAwere then reverse transcribed using the SMARTer Kit (Takara) in which the oligodT(30) 3' SMART CDS Primer II A was replaced by the oligodA(30) 3' SMART CDS Primer II A. The obtained full-length cDNAs were then amplified by PCR (14 cycles) using GXL PrimeSTAR DNA polymerase (Takara). cDNA size fractionation was then performed on 0.75% agarose gels (without ethidium bromide) to individualize three cDNA fractions: [0.3 - 2 kb], [2 – 5 kb], and [> 5 kb]. Dedicated biotinylated riboprobes, designed against the 5' 400 bp sequences of the 8,744 recent L1 (myBaits, Arbor Biosciences), were used to perform three independent captures of each cDNA fraction according to the supplier's recommendations (except for the addition of Block C solution = human Cot-1 DNA). Captured cDNAs were then amplified by PCR using Prime StarGXL DNA polymerase (Takara) with 10, 12 and 14 cycles respectively for the  <2 kb, 2-5 kb and >5 kb cDNA fractions. PCR products were pooled and purified using 0.55X AMPurePB beads (PacBio). Finaly, the three samples were pooled together and handled for SMRTbell template preparation and sequencing (Sequel II, Gentyane platform).