Illumina HiSeq 2500 sequencing; Diversity of early astrocyte reactive profiles in familial amyotrophic lateral sclerosis
Diversity of early astrocyte reactive profiles in familial amyotrophic lateral sclerosis
All cell cultures were maintained at 37°C and 5% carbon dioxide. iPSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies), and passaged using EDTA Generation of hiPSC-derived astrocytes was carried out using a previously described protocol 2. Briefly, after neural conversion (7 days in a chemically defined medium containing 1 μM Dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience) and 3.3 μM CHIR99021 (Miltenyi Biotec) neural precursors were patterned for 7 days with 0.5 μM retinoic acid and 1 μM purmorphamine, followed by a 4-day treatment with 0.1 μM purmorphamine. After a propagation phase (>60 days) with 10 ng/ml FGF-2 (Peprotech) and were terminally differentiated to astrocytes in presence of BMP4 (10 ng/ml, R&D) and LIF (10 ng/ml, Sigma-Aldrich).  The Promega Maxwell RSC simplyRNA cells kit including DNase treatment, alongside the Maxwell RSC instrument, was used for RNA extractions. The nanodrop was used to assess RNA concentration and the 260/280 ratio.  Library preparation followed the Illumina TruSeq RNA Access library preparation kit as per the manufacturer's instructions; 100ng total RNA was first fragmented, cDNA generated using random priming during first and second strand synthesis; sequencing adaptors are ligated to the resulting double-stranded cDNA fragments. The coding regions of the transcriptome were then captured from this library using sequence-specific probes before a final round of PCR amplification and second strand digestion occurs to create the final library. This strand-specific protocol was used for library preparation and samples were barcoded and multiplexed prior to sequencing.