organism: Mus musculus
description: Protocols: 46C mouse embryonic stem cells (derived from E14tg2a cell line; ESC) were grown at 37degreesC in a 5percent (v/v) CO2 incubator in Glasgow Modified Eagles Medium (GMEM, Invitrogen), supplemented with 10 percent (v/v) fetal bovine serum (FBS) (Sigma), 2 ng/ml of in-house synthesized LIF, 1 mM 2-mercaptoethanol, Non-Essential Amino Acids (Gibco) and Na-pyruvate (Gibco), on gelatin-coated (0.1percent (v/v)) dishes. We used Accutase (Sigma) for cell dissociation (which we inactivated with cell culture medium according to the manufacturers recommendations). Cells were passaged every other day at a seeding density of 3 million cells for a 10-cm petri dish. Medium was exchanged every day. The ESCs were differentiatied to Neural Stem Cellss (NSC). Briefly, 46C ES cells were plated at the density of 2 million cells on gelatin-coated 10 cm petri dish in 50percent Dulbeco Modified Eagles Medium, 50percent F12 (DMEM/F12, Invitrogen) medium supplemented with N2 and B27 (Gibco), BSA (Gibco), Non-Essential Amino Acids (Gibco), glucose (final concentration of 0.03 M), HEPES (final concentration of 4.5 mM) and 0.1mM beta-mercaptoethanol (Differentiation medium). Medium was exchanged after 24 and 48 hours and the cultures grown for additional 72 hours. Cells were then gently dissociated using Accutase (Sigma), the GFP+ cell fraction (corresponding to c.a. 70 percent of cells) was FACSorted and seeded onto laminin (Sigma) coated 75 cm2 Nunc flask (final concentration of laminin 10 mg/cm2 of culture surface, coating time - minimum 4 hours at 37 degrees C). Subsequently, cells were grown in differentiation medium supplemented with in-house prepared recombinant murine EGF and bFGF (both at concentration of 10 mg/ml) until loss of GFP expression and uniform up-regulation of Nestin expression. Cells where passaged when reaching 80 percent confluence, medium as exchanged every day. Cells from confluent culture dishes were dissociated with Accutase (Sigma), washed two times with full medium and lysed in Trizol reagent (Invitrogen, 500 ul of Trizol reagent per 5 million cell pellet) and snap frozen. At the day of RNA isolation, cell-Trizol mixes were thawed for 10 minutes and RNA isolation performed according to manufacturers recommendations. Isolated RNA samples were inclubated with TURBO DNase (Ambion) according to manufacturers recommendations. RNA quality was assessed with an Agilent 2100 Bioanalyzer. Samples with RNA Integrity Number (RIN) larger than 9 were included for subsequent RNA-seq experiments. The libraries were prepared using a standard TruSeq protocol, using TruSeq Stranded mRNA Sample Preparation kit. Briefly, 3ug of total RNA was used per sample. RNA was re-suspended in 50 ul of nuclease free water and mixed with 50 ul of oligo-dT beads to bind the poly-A RNA. The RNA-bead suspension was mixed thoroughly by pipeting 6 times. The suspension was incubated at 65 degrees for 5 minutes and slowly cooled down to 4 degrees. Beands with the bound poly-A RNA were isolated with magnet. Beads were washed with Wasing Buffer by gentile pipeting beads with the wasing buffer 6 times. The samples were next incubated on the magnetic stand for 5 minutes, the supernatant discarded and 50 ul of elution buffer were added to the sample. The entire mix was pipeted six times, vial closed and incubated at the thermal cycler at 80 degrees for 2 minutes. The mix was chilled to 25 degrees. 50 ul of the Bead Binding buffer was add to each sample and mixed by pipeting as above. The mix was incubated for 5 minutes and beads isolated using the magnetic stand. Beads were washed as above with the wash buffer and left on the magnet for 5 minutes.The supernatant was discarder and the beads mixed with 19.5 ul of the Fragment, Prime, Finish Mix. The entire volume was gently mixed and the samples sealed and incubated on the thermal cycler for 8 minutes at 98 degrees and immediately chilled to 4 degrees. Samples were then centrifuged briefly, and the first strand cDNA was synthetised. The bead mix was placed on the magnetic stand for 5 minutes, 17 ul of the supernatant (containing the fragmented and primed mRNA) was pipeted to a new 0.3 ml PCR tube and 8 ul of the first strand synthesis ActD, superscript II mix was add to the vial, the tube content was mixed and centrifuged. The sample was incubated at 25 degrees for 10 min, 42 degrees for 15 minutes, at 70 degrees for 15 minutes and chilled to 4 degrees. To generate second strand: 5Ul of the resuspension buffer form the kit was added and then 20 ul of the thawed Second Strand Marking Master Mix and mixed with the solution. The samples were incubated at 16 degrees for 1 hour. The samples were brought to room temperature and the cDNA isolated with 90 ul of the Ampure XP beads. 80 percent ethanol solution was used in the washing steps of the Ampure isolation procedure. Beads were resuspended in 17.5 ul of the resuspension buffer and incubated for 2 minutes at room temp. 15 ul of the supernatant was transfered to a new 0.3 ml PCR tube. Then 2.5 ul of the resuspension buffer was added to the sample, the whole sample was mixed with 12.5 ul of the A-tailing Mix and incubated at 37 degrees for 30 minutes, 70 degrees for 5 minutes and held at 4 degrees. Immediately after, 2.5 ul of the resuspension buffer was added to the sample along withand 2.5 Ligation mix and 2.5 of RNA-Adapter mix. The samples were next centrifuged (280g for 1 min) and incubated at 30 degrees for 10 minutes. 5Ul of the Stop Ligation Buffer was added and mixed with the sample. CDNA was isolated using 42ul of Ampure Beads XP accoring to the TruSeq protocol. Samples were eluted with 22.5 ul of Resuspension buffer. 20 ul of the solution wasa trnsferred to a nw 0.3 ml PCR tube. 5ul of thawed PCR primer coctail and 25 ul of PCR master mix were added and Pcr reaction was performed (initail 98 degrees 30 sec followed by 15 cycles of 98 degrees for 10 sec, 60 degrees for 30 sec, 72 degrees for 30 sec, and a final incubation at 72 degrees for 5 min). The samples were than isolated with the Ampure Beads accoring to the TruSeq protocol.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-09-01T17:02:54Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:54:27Z
attributes.tag: External Id
attributes.value: SAMEA2259740
attributes.tag: INSDC center name
attributes.value: European Molecualr Biology Laboratory, Genome Biology Unit
attributes.tag: INSDC first public
attributes.value: 2018-09-01T17:02:54Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:54:27Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2125:Embryonic Stem Cell Biological Replicate 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: ES-E14 cell
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: sample name
attributes.value: E-MTAB-2125:Embryonic Stem Cell Biological Replicate 1
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: specimen with known storage state
attributes.value: fresh specimen