Illumina Genome Analyzer IIx paired end sequencing; Regulation of the ESC transcriptome by nuclear long non-coding RNAs
Regulation of the ESC transcriptome by nuclear long non-coding RNAs
Nuclear RNA fractions were obtained by resuspending cells to 2x107 / ml in ice-cold nuclei buffer (10 mM Tris pH 7.6, 10 mM NaCl, 2 mM MgCl2) supplemented with protease inhibitor cocktail and RNAse inhibitor. Cells were kept on ice for 10 minutes for hypotonic swelling prior to adding an equal volume of nuclei buffer containing 0.5% NP-40 for 5 minutes. Nuclei were collected, washed in nuclei buffer / 0.5% NP-40 and lysed in TRIzol. ESC colonies were maintained on gelatinized cell culture dishes, on a feeder layer of irradiated mouse embryonic fibroblasts in knock-out DMEM supplemented with 15 % fetal bovine serum, non-essential amino acids and 1,000 U/ml leukemia inhibiting factor. NPCs were maintained on gelatinized cell culture dishes in N2 expansion medium composed of 50:50 DMEM / F12: neurobasal medium, supplemented with 1x N2, 0.05x B-27, 50 ug /ml BSA (fraction V), 1ug / ml laminin, and 10 ng / ml each murine basic fibroblast growth factor and epidermal growth factor Total RNA was isolated using TRIzol reagent. For ASO knock-down studies, total RNA was collected using TRIzol and precipitated in the presence of 7.5 ug GlycoBlue (Ambion, Life Technologies). RNA integrity was assessed on the Agilent 2100 Bioanalyzer instrument and only RNA samples with integrity scores > 9.5 (median 10.0) were used. Stranded libraries for paired-end sequencing were prepared as described in Parkhomchuk, D., Borodina, T., Amstislavskiy, V., Banaru, M., Hallen, L., Krobitsch, S., Lehrach, H., and Soldatov, A. (2009). Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 37, e123.
CSHL
E-MTAB-3198:AB2.2 NPC nuclear polyA
Cold Spring Harbor Laboratory
E-MTAB-3198:AB2.2 NPC nuclear polyA
Experimental Factor: cell type
neural progenitor cell
Experimental Factor: cellular component
nucleus
Experimental Factor: compound
none
LIBRARY_STRAND
first strand