title: GSM2668096: Mpeg 1 days post injury sham1  ; Danio rerio; RNA-Seq
library_construction_protocol: For nuclei isolation, hearts were dissected at appropriate time points and  washed in hypotonic buffer H (20 mM HEPES (pH 7.9), 15 mM MgCl­2, 10 mM KCl, 1 mM DTT, and 1 X Complete protease inhibitor) and subsequently resuspended in 500 μL of buffer H. Embryos were transferred to a Dounce homogenizer and dissociated by 10 strokes with the loose fitting pestal A and incubated on ice for 5 minutes. Further dissociation was carried out by 10 strokes with tight fitting pestal B, every 5 minutes for 15 minutes. Nuclei were collected by centrifugation (2000 g, 4 °C) and resuspended in 1 mL buffer NDP (10 mM HEPES (pH 7.9), 40 mM NaCl, 90 mM KCl, 0.5 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 1 mM dithiothreitol and 1 X Complete protease inhibitor). For nuclei purification, nuclei were incubated with 5 mg of M-280 streptavidin-coated dynabeads with rotation for 30 minutes at 4 °C. A flow-based system based on the previously published protocol by was used to capture the nuclei bound on the streptavidin beads. A 10 mL seriological pipette (VWR, cat # 8913-898) attached to a 1 mL micropipette tip (Rainin reach pipet tip, cat # RT-L1000S), both pre-treated with NPB/BSA for 30 minutes, and added to a MiniMACS separator magnet (OctoMACS Separator, Miltenyl Biotec, cat # 130-042-109). A two-way stopcock (Biorad cat # 732-8102) was connected to the end of the 1mL micropipette tip via a piece of Tygon tubing (Fisher Scientific, cat # 8220) and the flow-rate set to ~0.75 mL/min. The nuclei beads suspension was diluted by addition of 9 mLs of nuclei pulldown buffer with 0.01 % Triton X-100 (NPBt) and added to the slow-flow setup. The tip was subsequently removed from the stand and the nuclei-beads released from the tip by slowly pipetting 1 mL of NPBt in and out of the tip. The solution was then diluted again to 10 mL with NPBt and added again to the slow-flow setup. Nuclei-beads were eluted in 1 mL of NPBt as described above and the NPBt removed using a magnetic stand (DynaMag TM-2 magnet, Invitrogen, cat # 12321D). Nuclei-beads were then processed for RNA extraction. RNA extraction and DNase treatment were carried out using the RNAqueous®-Micro Kit (Life Technologies, cat # AM1931). RNA integrity was checked with a RNA pico chip (Agilent Technologies, cat # 5067-1513) using the Agilent 2100 Bioanalyzer. cDNA was synthesized and amplified from 100 pg – 1 ng of input RNA using SMART-seqTMv4 ultra low input kit for RNA (Clontech laboratories, cat #’s 634888, 634889, 634890, 634891, 634892, 634893, and 634894). Sequencing libraries were prepared using the Nextera XT DNA library preparation kit and sequencing performed on a NextSeq platform using a NextSeq™ 500 High Output Kit for the generation of 80 base pair paired end reads (150 cycles) (Illumina, cat # FC-404-1002).
attributes.tag: GEO Accession
attributes.value: GSM2668096