Illumina HiSeq 2000 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
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 For transfection of ASOs using nucleofection technology (Lonza), ESCs were harvested following soaking off of feeder cells for one hour, washed in D-PBS and passed through a 70 um nylon cell strainer. Cell count and viability was determined by trypan blue staining on a Countess automated cell counter. For each reaction, 5x104 viable cells were resuspended in P3 Primary Cell solution (Lonza), mixed with 2 uM control or 2 uM target-specific ASO and transferred to nucleocuvettes for nucleofection on a 4D-Nucleofector System (Lonza) using program code "DC-100". Cells were subsequently transferred onto gelatinized cell culture plates containing pre-warmed and supplemented growth medium. Growth medium was changed once after 16 hours. 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. Libraries were prepared from poly(A)+ selected RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the manufacturers instructions, except that final libraries were subject to size selection followed by gel purification and validation on the Agilent 2100 Bioanalyzer.
CSHL
E-MTAB-3198:ESC Firre ASO#2 Rep4
Cold Spring Harbor Laboratory
E-MTAB-3198:ESC Firre ASO#2 Rep4
Experimental Factor: cell type
embryonic stem cell
Experimental Factor: cellular component
whole cell
Experimental Factor: compound
2'-O-Methoxyethyl Antisense Gapmer (Firre antisense, oligo #2)