GSM6339166: Sample 1 Patient 187 [FLU013014]; Homo sapiens; RNA-Seq
Co-Isolation protocol was performed to extract at the same time DNA and PolyA RNA from the same sample. Poly-A RNA was selected with Dynabeads Oligo (dT) (Ambion) and reverse-transcribed with AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) using poly-dT oligos carrying a 7 bp-index. Up to 8 samples with similar overall RNA content were pooled together and subjected to linear amplification by IVT using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). Next, aRNA was fragmented into 250-350 bp fragments with RNA Fragmentation Reagents (Ambion) and dephosphorylated with FastAP (Thermo) following manufacturer´s instructions. Partial Illumina adaptor sequences (Jaitin, 2014) were ligated with T4 RNA Ligase 1 (New England Biolabs), followed by a second RT reaction. Full Illumina adaptor sequences were added during final library amplification with KAPA HiFi DNA Polymerase (Kapa Biosystems). RNA-seq libraries quality controls consisted in quantification with Qubit 3.0 Fluorometer (Life Technologies) and size profiles examination with Agilent's 4200 TapeStation System.