Protocols: Pmi28 myoblasts were washed with PBS, detached using 0.25 % Trypsin/1 mM EDTA for 3 min and collected in a centrifuge tube. After centrifugation for 4 min at 1300 g, cells were washed with PBS and centrifuged again. For native ChIP-seq, the pellet obtained from three 150 mm cell culture plates roughly corresponding to 15 Mio myoblasts was directly snap frozen in liquid nitrogen and kept at -80º C. For Hi-C, cells were re-suspended in medium to a final volume of 10 ml in 15 ml falcon tubes. 666 μl of 16% formaldehyde were added to the tube (resulting in a final concentration of 1% formaldehyde ); the tube was briefly shaken and then rotated for exactly 10 min at 21º C. To quench the reaction, 593 µl of 2.5 M glycine were added; the tube was briefly shaken and left rotating for 5 min. After quenching, tubes were centrifuged for 5 min at 2,000 g and the supernatant was discarded. Cells were resuspended in 1.5 ml PBS, transferred into 2 ml tubes, centrifuged at 2,000 g for 5 min at RT and the supernatant was discarded. The pellets were snap-frozen in liquid nitrogen and kept at -80º C. Myotube samples were collected as follows in order to separate differentiated myotubes from undifferentiated myoblasts: first, myotubes were gently trypsinized using 0.025 % Trypsin/0.1 mM EDTA; when myotubes detached, leaving the majority of myoblasts still attached to the bottom of the dish, the supernatant containing myotubes and a proportion of myoblasts was transferred to a fresh 150 mm cell culture dish. The remaining myoblasts were allowed to re-adhere to the dish bottom, whereas myotubes did not re-adhere and thus could be collected with the supernatant. After centrifugation for 4 min at 1300 g, cells were washed with PBS and centrifuged again. For native ChIP-seq, the pellet obtained from three 150 mm cell culture plates roughly corresponding to 10 Mio myotubes was snap frozen in liquid nitrogen and kept at -80º C. For Hi-C, myotubes were fixed, quenched and frozen in the same way as myoblasts. We estimated that our myotube samples contained 10-20% of myoblasts. Mouse myoblast cell line were grown in Nutrient Mixture F-10 Ham supplemented with 20% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin at 37°C and 5% CO2. For differentiation, cells were seeded at a density of 8 x 104 cells/cm2 and incubated in differentiation medium (high glucose DMEM supplemented with 1% Penicillin/Streptomycin and 2% horse serum) at 37°C and 5% CO2. Differentiation medium was replaced every 2 days. Typically, after 3-4 days a sufficient number of myotubes formed and after 5-6 days they start to contract. The following nChIP-seq protocol was adapted from (Punzeler et al., 2017) and (Link et al., 2018). Immunoprecipitations were carried out with S1 mononucleosomes derived from Pmi28 myoblasts and Pmi28 myotubes. nChIP-seq experiments were performed in independent duplicates. Per immunoprecipitation, 5 Mio cells were used. Cells were resuspended in 750 µl PBS + 1 x PI + 0.3 % Triton X-100 and incubated for 10 min at 4 °C while rotating. The cell lysate was centrifuged for 10 min at 3,000 g and 4°C, the pellet was resuspended in 625 µl PBS + PI and centrifuged for 5 min at 3,000 g at 4°C. Supernatant was carefully removed and the pellet was resuspended in 62.5 µl EX100 (10 mM HEPES pH 7.6, 100 mM NaCl, 1.5 mM MgCl2, 0.5 mM EGTA, 10 % (v/v) glycerol, 10 mM β-glycerol phosphate) + 1x PI + 1 mM DTT and transferred into a DNA low binding tube. CaCl2 concentration was adjusted to 2 mM and 0.1875 µl MNase (1 U/µl) were added. MNase digest was carried out for 20 min at 26 °C. The reaction was stopped by the addition of EDTA to a final concentration of 10 mM and the suspension was centrifuged at 13,300 rpm for 30 min at 4 °C in a tabletop centrifuge. The supernatant (S1 mononucleosomes) was transferred into a fresh DNA low binding tube and filled up to 500 µl with EX100 + 1x PI + 1 mM DTT. 25 µl (5 %) were saved as input. 5 µg of antibody (anti-RNA polymerase II CTD repeat YSPTSPS, 8WG16, Abcam ab817) were added per IP (5 Mio cells) in 500 µl and incubated with mononucleosomes at 4 °C overnight while rotating. On the next day, magnetic beads (Dynabeads Protein G, Thermo Fisher) were washed 3 times with EX100 + 1 x PI + 1 mM DTT (500 µl/wash), resuspended in 30 µl EX100 + 1 x PI + 1 mM DTT per IP, added to the antibody-mononucleosome suspension and rotated at 4 °C for 3 h. A beads-only IP served as negative control. Washing steps were conducted using 500 µl of buffer per washing step and samples were rotated at 4 °C for 5 min. Beads were magnetically separated after each washing step. Beads were washed twice with WB1 (10 mM Tris pH 7.5, 1 mM EDTA, 0.1 % SDS, 0.1 % sodiumdeoxycholate, 1 % Triton X-100), twice with WB2 (10 mM Tris pH 7.5, 1 mM EDTA, 0.1 % SDS, 0.1 % sodiumdeoxycholate, 1 % Triton X-100, 150 mM NaCl), once with TE + 0.2 % Triton-X 100 and once with TE buffer. Washed beads were resuspended in 100 µl TE buffer. To the input DNA sample, 75 µl TE were added. 3 µl of 10 % SDS and 5 µl of proteinase K were added and the suspensions were incubated 1 h at 65 °C. After brief vortexing, suspensions were magnetically separated and supernatant was transferred into DNA low binding tubes (Eppendorf). Beads were washed with 100 µl TE + 0.5 M NaCl, vortexed briefly, magnetically separated and the supernatant added to the first supernatant. For input DNA, 100 µl TE + 0.5 M NaCl were added. Eluted IP DNA and input DNA were Phenol/Chloroform/Isoamylalcohol extracted using MaXtract high density tubes (Qiagen) and ethanol precipitated according to a standard protocol. After extraction, the IP DNA pellets were resuspended in 12 µl 10 mM Tris-HCl pH 7.5 and the input DNA pellet was resuspended in 20 µl 10 mM Tris-HCl pH 7.5. DNA concentrations were determined via the Qubit dsDNA High Sensitivity Kit (Invitrogen) and DNA size was analyzed on a DNA 1000 BioAnalyzer chip (Agilent Technologies). Illumina sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and the NEBNext Multiplex Oligos for Illumina (New England Biolabs) according to the manufacturer's instructions. The number of amplification cycles was scaled according to the amount of input material, amplification was validated by measuring DNA concentrations using the Qubit dsDNA High Sensitivity Kit (Invitrogen) and library quality was determined using a Bioanalyzer (Agilent Technologies) and a DNA 1000 Bioanalyzer chip (Agilent Technologies).
ENA first public
2021-12-06
ENA last update
2021-12-06
External Id
SAMEA6829403
INSDC center alias
Ludwig-Maximilians University
INSDC center name
Ludwig-Maximilians University
INSDC first public
2021-12-06T12:10:44Z
INSDC last update
2021-12-06T12:10:44Z
INSDC status
public
Submitter Id
E-MTAB-9060:blasts_input_r1
broker name
ArrayExpress
cell line
Pmi28
cell type
myoblast
common name
house mouse
developmental stage
adult
disease
normal
genotype
wild type genotype
organism part
skeletal muscle tissue
sample name
E-MTAB-9060:blasts_input_r1