title: GSM1701699: genomic DNA; Homo sapiens; OTHER
library_construction_protocol: Preparation and amplification of nucleic acids were performed with a customized version of the μMACSSuperAmpKit (MiltenyiBiotec); if not mentioned explicitly, procedures were according to the manufacturer protocol. The protocol is based on magnetic coupling of mRNA and retaining the nucleic acid in low volume flow-through columns for greatly simplified handling. For selective mRNA isolation, magnetic micro beads coupled to oligo-dT primers were applied. To retain the genomic DNA the eluates from the first two washing steps after loading the cell lysate onto the column were collected into a 1.5 mL reaction tube for later DNA precipitation and whole genome amplification. On column cDNA synthesis was performed at 42°C for 60 min according to the following protocol by applying the total reaction master mix onto the column: 20 µL contained 2 µL 10x Reverse Transcriptase Buffer (Ambion), 0.5 mM dNTPs, 1µg T4 Gene 32 Protein (NEB), 400 U M-MLV Reverse Transcriptase (Enzymatics), 20 U RNase Inhibitor (Ambion). After collection of magnetic beads containing synthesized cDNA by centrifugation and 3’-tailing according to the manufacturer, PCR amplification was performed. To the 3’-tailing reaction, in total 30 µL, the following PCR master mix was added: 76.5 µL PCR master mix  contained 14 µL 5x PhusionHF buffer (Finnzymes), 0.5 mMdNTPs, 60 µL resuspended μMACSSuperAmp PCR mix, 2 U PhusionTaq (Finnzymes); the following cycling conditions were applied on a PTC-200 (MJ Research) thermal cycler: 78°C for 30 sec, 95°C for 1 min, [98°C for 3 sec, 64°C for 30 sec, 72°C for 2 min]x 40 cycles, 72°C for 5 min. Amplification of genomic DNA was performed with the REPLI-g Midi Kit (Qiagen) for 16 hours at 30° C according to the manufacturer’s recommendations. Before whole genome amplification, DNA was ethanol precipitated by adding 0.1 volumes of 3M sodium acetate solution and 5 µg glycogen (Ambion) to 1 volume of DNA sample. After precipitation the pellet was resuspended in 10 µL of Elution Buffer (Qiagen).
identifiers.namespace: GEO
identifiers.id: GSM1701699