title: GSM9567302: Patient 8, TBM, CSF, D28; Homo sapiens; RNA-Seq
library_construction_protocol: CSF was collected directly into PAXgene RNA tubes (QIAGEN/BD Biosciences) at the bedside from patients enrolled in the LASER-TBM and INTENSE-TBM trials. Lumbar puncture was done at day 3 (LASER-TBM) and day 7 (INTENSE-TBM) and week 4 (both trials) per the study protocols. Total RNA was extracted using the PAXgene Blood RNA Kit (QIAGEN, Venlo, Netherlands). RNA extraction of samples from the two trials was done at separate timepoints but DNA libraries were prepared at the same time. Samples were stored at -80˚C before and after RNA extraction. The quantity and quality of RNA was assessed using a Qubit fluorimeter (ThermoFisher) and Agilent 2100 Bioanalyzer. The Watchmaker DNA library prep kit (Watchmaker Genomics, Boulder, USA, CAT# 7K0102) with ribosomal depletion was used to construct libraries. CSF samples were divided into three batches of different input concentrations (2 ng, 1 ng, 0.5 ng) so that RNA loading could be maximised for samples with higher RNA concentrations. Following library preparation, DNA library size distribution was assessed using the D1000 TapeStation kit processed on the 4200 TapeStation (Agilent Technologies). Concentration was measured using the QuantiFluor dsDNA System (Promega Corporation). Libraries were sequenced on an Illumina NovaSeq 6000 with a paired-end 100 cycle read configuration with a target sequencing depth of 30 million reads/sample. Five rounds of re-pooling and sequencing were done, which achieved a median read depth of 41 million reads/sample (range 16.2-149 million).