title: GSM1977035: 60_sALS; Homo sapiens; RNA-Seq
library_construction_protocol: Laser capture microdissection was done according to the protocol described previously (Rabin et al). Briefly, Thirty-five to 50 sections of frozen OCT-embedded human lumbar spinal cord were cut at a thickness of 9 µm in a -18C cryotome and placed onto uncharged glass slides. The sections were returned immediately to -78C after production, and maintained at that temperature for a minimum of three hours. Staining with cresyl violet acetate was accomplished in a 10-step, timed, nuclease-free immersion process. Motor neurons were microdissected from each slide using a Pixcell IIe Laser Capture Microdissection (LCM) System (Arcturus Bioscience) and CapSureTM Macro LCM Caps (Applied Biosystems). Each LCM session had an upper time limit of 2.5 hours. In addition to the motor neuron enriched LCM material, the remaining anterior horn region was collected to create a second parallel pool. RNA isolation was carried out in each using an RNeasy Micro kit (Qiagen), which employs guanadinium isothiocyanate and 2-mercaptoethanol extraction and column purification, including an on-column DNase I digestion step. Aliquots of each RNA pool were analyzed for quality and quantity on a Bioanalyzer RNA Pico chip (Agilent) and only those samples with RNA Integrity Numbers (RINs) > 5 or evidence of 28S peaks on the electropherogram tracings were advanced to the next step. Total RNA (10 ng) was amplified to cDNA using random priming with the Ovation® RNA-Seq System (NuGEN).  This procedure involved initial generation of double-stranded cDNA followed by amplification through production of single-stranded cDNA using the SPIA® process.   The single-stranded cDNA was then copied into double-stranded cDNA and quantified using the PicoGreen kit (Invitrogen) assayed with the FUSION system (Packard Biosciences), resulting in a total yield of 3-4 ug amplified cDNA’s.  Standard concentration curves using bacteriophage lambda DNA were generated for each PicoGreen analysis, and the samples were diluted by 10-fold serial dilutions.  QC of the amplified cDNA was determined using a Bioanalyzer running an RNA 6000 Nano LabChip (Agilent).  Double-stranded cDNA (1-2 ug) was fragmented to 150-200 bp sizes using Adaptive Focused AcousticsTM (Covaris, Inc., Woburn, MA), and QC of the fragmented cDNA was performed using a Bioanalyzer DNA Chip 1000 (Agilent).   Fragmented cDNA’s were concentrated using the QIAquick PCR Purification Kit (Qiagen) and 200 ng of the fragmented cDNA’s (determined following PicoGreen quantitation) were end-repaired, followed by adaptor ligation onto the fragments and amplification using the Encore® NGS Library System I (NuGEN).  Library QC was performed using a Bioanalyzer DNA Chip 1000.
attributes.tag: GEO Accession
attributes.value: GSM1977035