miR-seq GBM frontal lobe donor
Glioblastoma Multiforme (GBM) (IV grade) sample was obtained from the frontal lobe of a male patient. The sample was dissected from the proliferative core of the tumor and stored at −80°C for molecular studies. miRVana miRNA Isolation Kit (Ambion, USA) was used following the manufacturing instruction to isolate miRNA from this sample. GBM sample was collected during neurosurgery of the patient at the Policlinico Gemelli in Rome. The study was approved by the local ethical committee on the use of human samples for experimental studies.   MiRNA capturing and library construction were conducted using Illumina's TruSeq Small RNA Sample Prep Kit following the manufacture protocol. Briefly, 3' adapter was ligated to total RNA using truncated T4 RNA Ligase 2 without ATP, then 5' adapter was added using T4 RNA Ligase with ATP. Afterwards, reverse transcription followed by PCR was used to create cDNA constructs based on the small RNA ligated with 3' and 5' adapters. The introduction of index sequence (bar-codes) was done at the PCR step. Finally, gel electrophoresis was used to purify the amplified cDNA construct in preparation for subsequent cluster generation. After library quality check by Bioanalyzer High Sensitivity chip, clusters were generated and libraries were run on HiSeq2000 by using standard Illumina sequencing workflow with the multiplexing option. The mature miRNA library from the GBM frontal lobe sample was sequenced on two lanes of Illumina HiSeq2000 instrument following the manufacture protocol with three different bar-codes. Primary data analyses of sequence data and quality controls were performed using the Illumina pipeline (version 1.7). The reads were filtered using Illumina's HiSeq2000 softwares, Illumina adapter removal and indexes separation was also performed.