title: GSM2805183: Eed null mESC Bru-seq rep1; Mus musculus; RNA-Seq
library_construction_protocol: Experiments were carried out on mouse ES cells gown on feeder-free, 1% gelatinized 100mm tissue culture dishes. 5-Bromouridine (BrU) was added to cells at a final concentration of 2mM (from a100mM BrU stock) and incubated in the incubator for 10 min. Media was aspirated off and cells were lysed in 4ml of Trizol. 800µl of chloroform was next added to the lysed cells in 5ml epp tubes, vortexed and spun down at 5200 g for 15min. Equal volume of isopropanol was added to the aqueous phase, mixed, left at RT for 5-10 min and spun down at 4oC for 20min at max speed (14000 g) in a tabletop centrifuge to precipitate the RNA.  Resulting RNA pellet was washed twice with 70% EtOH, air dried and resuspended in 200-400 µl Nuclease-free water. The amount of RNA was quantified using Nanodrop, and a small fraction of the total RNA was run on agarose gel to determine the quality of purified total RNA. Bru-RNA enrichment: Mouse aBrdU antibody (SCBT, sc-32323) was coupled to Dynabeads M-280 Sheep Anti-Mouse IgG (11201D). 1ml of magnetic beads was washed 4 x with PBS-DEPC/0.1%BSA and 500ul of Ab  was incubated with 1ml of beads and yeast RNA (500ng/µl final concentration) for 2-3 hours at 4oC, washed 3x with PBS-DEPC/0.1%BSA/Tween 0.05%.The final volume was raised to 1ml. For enrichment of Bru-RNA, 25-50µg of total RNA (after preheating at 95oC and immediate cooling) was added to 50µl of Antibody-bound beads in 1 ml of PBS-DEPC/0.1%BSA/Tween 0.05% and incubated for 90 min at RT with a foil wrapped around the tube. Beads were washed 4x with PBS-DEPC/0.1%BSA/Tween 0.05%, 5-10min (last wash was done with Tween-free buffer). Enriched RNA was either eluted by addition of 25µl of DEPC-water and incubation at 95oC for 10min with occasional flicking/shaking, or Trizol extraction. The enriched RNA was depleted of ribosomal RNA and used for generation of RNA-seq library.
attributes.tag: GEO Accession
attributes.value: GSM2805183