HUDEP2-OE-COUPA-2
Homo sapiens
Protocols: HUDEP1 and HUDEP2 cells were grown according to standard protocols. CUT&RUN was performed according to the LoV-U protocol described in (Zambanini et al., 2022). Per each sample 500,000 cells were harvested (HUDEP1/2 WT and COUPTFII-expressing). Cells were washed three times in Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free), then resuspended in 40 μl NE per sample and bound to 20 μl Magnetic ConA Agarose beads (ABIN6952467) equilibrated in binding buffer (HEPES pH-7.5 [20 mM], KCl [10 mM], CaCl2 [1 mM], MnCl2 [1 mM]). After incubation, nuclei and beads were resuspended for 5 minutes in EDTA wash buffer (HEPES pH-7.5 [20 mM], NaCl [150 mM], spermidine [0.5 mM] in Roche Complete Protease Inhibitor EDTA-Free (COEDTAFRO), + EDTA [0.2 mM]). Samples were divided in 200 μl PCR tubes and antibody incubation was performed in 200 μl wash buffer with 2 μl of the right antibody (List of sample and Ab at the end of the paragraph) overnight (ON) at 4 °C on a rotator. After ON incubation, samples were washed five times in wash buffer and resuspended in 200 μl of pAG-MN buffer (wash buffer supplemented with pAG-MNase [0.6 μg/ml]) for 30 minutes at4 °C on a rotator. pAG/MNase was a gift from Steven Henikoff (Addgene #123461), expressed and purified as described previously (Meers et al., 2019a). Samples were washed five times, followed by digestion for 30 minutes in wet ice in 50 µl wash buffer with 2 mM CaCl2. After 30 minutes, the digestion buffer was moved to new collection tubes and the reaction on the beads was stopped with 50 μl of 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.5 M]) and the samples were incubated for 1 hour at 4 °C. Beads were collected on the magnetic rack, while the supernatant was mixed in the corresponding collection tube, where it was cleaned up twice using Mag-Bind TotalPure NGS beads (Cat. #M1327, Omega Bio- Tek) at 2X, and then resuspended in 20 μl Tris-HCl pH 7.5. Library preparation was performed using the KAPA Hyper Prep Kit for Illumina platforms (Cat. #KK8504, KAPA Biosystems) according to the manufacturer's guidelines with the following modifications. End repair and A-tailing was performed with 0.4x reactions with 20 μl of purified DNA. The thermocycler conditions were set to 12 °C for 15 min, 37 °C for 15 min and 58 °C for 25 min to prevent thermal degradation of the shortest fragments. Adapter ligation was done with 0.4x reactions. KAPA Dual Indexed adapters were used at 0.15 μM. A post-ligation clean-up was performed with Mag-Bind TotalPure NGS beads at 1.2x. Resuspension was done in 10 mM Tris-HCl pH 8.0. Library amplification was performed with 0.5x reactions. The thermocycler was set with the following conditions: initial denaturation at 98 °C for 45 sec, denaturation at 98 °C for 15 sec, annealing/elongation at 60 °C for 10 sec, final extension at 72 °C for 1 min, hold at 4 °C, with 13 cycles. After amplification, a post-amplification cleanup was performed with 1.2x beads. Libraries were then run on an E-Gel EX 2% agarose gel (Cat. #G402022, Invitrogen) for 10 min using the E-Gel Power Snap Electrophoresis System (Invitrogen). Bands of interest between 150 and 500 bp were cut out and purified using the QIAquick Gel Extraction Kit (Cat. #28706, QIAGEN) according to manufacturer's instructions. Libraries were quantified with the Qubit (Thermo Scientific) using their high sensitivity DNA kit (Cat #Q32854, Thermo Scientific).
ENA first public
2025-10-30
INSDC center name
Wallenberg Centre for Molecular Medicine, Linköping University, Linköping, Sweden Division of Molecular Medicine and Virology, Department of Biomedical and Clinical Sciences, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden
INSDC status
public
Submitter Id
E-MTAB-14720:HUDEP2-OE-COUPA-2
broker name
ArrayExpress
cell_line
HUDEP2
cell_type
erythroid progenitor
collection date
not collected
common name
human
genotype
COUPTFII OE
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
umbilical cord
sample name
E-MTAB-14720:HUDEP2-OE-COUPA-2
scientific_name
Homo sapiens