title: Ion Torrent Proton sequencing; The Functional Analysis of Granulins and Progranulin
design: The Functional Analysis of Granulins and Progranulin
library_construction_protocol: SH-SY5Y human neuroblastoma cells (ATTC CRL-2266TM) were cultured in minimum essential medium (Life Technologies 21090-022) containing 10% heat-inactivated foetal bovine serum (Life Technologies 10270-106), 2mM L-Glutamine (Sigma) 100 g streptomycin/ml,100i Penicillin/ml (Sigma) and 1% Non-essential amino acids (Life Technologies 11140-035) and plated at 8-10 x103 per cm3 in T-75 flasks (Corning). After 24 hours growth in a gassing incubator at 37C, 5% CO2 the medium was changed to N2 (Dulbeccos Modified Essential Medium:F12 containing 15mM HEPES and L-Glutamine (Life Technologies 31330-038) containing 2mM L-Glutamine (Sigma), 100 g streptomycin/ml,100i Penicillin/ml (Sigma) 1% N2 100x (Life Technologies 17502-048) (final concentration) and 1 M all-trans retinoic acid (Sigma R2625) (1mM dissolved in 99% ethanol and used at 1:1000). After 72 hours, five confluent T75 cm2 flasks of semi-differentiated SH-SY5Y cells were pelleted and resuspended in 25mls of N2 medium containing 1 M retinoic acid (as above). I.0ml of the resulting cell suspension was then added to each of sixteen 10cm2 cultures dishes (Corning) each containing a further 8mls of N2 medium. Further differentiation was facilitated by media changes every 3-4 days until the cells were terminally differentiated after 3 weeks in culture. Cells were treated by replacing the medium in each dish with fresh medium (2.5mls/dish) containing either 0.5 g/ml progranulin or 50ng/ml of the individual granulins for 30min before cell harvest. Cells were harvested at the specific time points  by removing the medium, washing the cells briefly with ice-cold  DPBS (Sigma) and lysing by adding 1.050mls RLT buffer (Qiagen RNeasy Mini Kit (Cat No 74104)). The cell lysates  were lysed using spin columns (Qiagen Qiashredder kit Cat No 79654) and purified using the RNA Qiagen RNeasy Mini Kit (Cat No 74104). The quantities for each sample were measured using a Nanodrop 1000 Spectrophotometer and the quality of the RNA produced assessed by running the samples on an Agilent 2100 Bioanalyzer using Agilent RNA 6000 Nano Kit chips. The RIN number of each sample was above 7 for each sample.  Depletion of ribosomal RNA was carried out using the RibominusTM Eukaryote Kit (Ambion A15026), material was digested using RNase III and size selected using Agilent clean seq magnetic beads. Prior to library construction RNA fragement size was validated by running the samples on an Agilent 2100 Bioanalyzer.  Library construction was carried out using the Life technologies Ion Total RNA-Seq Kit v2.
attributes.tag: Experimental Factor: Paragranulin 0.5 g/m
attributes.value: growth condition