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 differentiated 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's 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. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Do PCR with the following setup: 98 degrees: 30 sec and 15 cycles of 98 degrees: 10 sec,60 degrees: 60 sec, 72 degrees: 30 sec, 72 degrees: 7 minute. Gel excise fragments of 400 nt and sequence by paired-end sequencing
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-09-01T17:02:54Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:53:20Z
attributes.tag: External Id
attributes.value: SAMEA2729829
attributes.tag: INSDC center name
attributes.value: EMBL GENOMICS CORE FACILITY, HEIDELBERG, GERMANY
attributes.tag: INSDC first public
attributes.value: 2018-09-01T17:02:54Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:53:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2063:Embryonic Stem Cell Biological Replicate 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: E14TG2a-derived
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: individual
attributes.value: ESC2
attributes.tag: sample name
attributes.value: E-MTAB-2063:Embryonic Stem Cell Biological Replicate 2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: specimen with known storage state
attributes.value: fresh specimen