GSM3677386: IgG_CUT&RUN_EILP_rep2_GB3051; Mus musculus; OTHER
Cells were fixed by adding 1:15 volume of 16%w/v formaldehyde solution (Thermo Fisher Scientific) and incubating at RT for 10 minutes. Reaction was terminated by adding 1:10 volume. 1ml RIPA buffer (10 mM Tris-Cl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.2% sodium dodecyl sulfate, 1% Triton X-100) added to each sorted 500 ALP, EILP, and ILCP cells, and incubated at room temperature for 30 minutes. Cells were rinsed with 500 μl binding buffer (10 mM Tris-Cl, 1 mM EDTA, 150 mM sodium chloride, 0.1% Triton X-100) twice and re-suspended in 50 μl binding buffer. To prepare TCF1 bounded PA-MNase (Ab+PA-MNase), TCF1 and the PA-MNase at a molecular ratio of 1:2 were pre-incubated at 4 °C for 30 minutes in 50 μl binding buffer. TCF1+PA-MNase were add to 50 μl binding buffer re-suspended cells and incubated one hour at 4 °C with rotation. Cells were washed using 200 μl wash buffer (10 mM Tris-Cl, 1 mM EDTA, 150 mM sodium chloride, 0.1%w/v SDS, 0.1%w/v sodium deoxycholate and 1%v/v Triton X-100) three times and pelleted by centrifugation at 600g for 2 minutes. Next, cells were rinsed using 200 μl rinsing buffer (10 mM Tris-Cl and 10 mM sodium chloride, 0.1%v/v Triton X-100). The MNase digestion was initiated by re-suspending the rinsed cells in 40 μl reaction solution buffer (10 mM Tris-Cl, 10 mM sodium chloride, 0.1%v/v Triton X-100, 2 mM CaCl2) and incubated at 37 °C for 3 minutes. The reaction was stopped by adding 80 μl stop buffer (20 mM Tris-Cl (pH 8.0), 10 mMethylenedioxy-bis-(ethylenenitrilo)-tetraacetic acid (EGTA), 20 mM sodium chloride, 0.2%w/v SDS) and 1 μl proteinase K (SigmaAldrich), then incubating at 65 °C for overnight. DNAs were purified by using phenol-chloroform extraction and ethanol precipitation. The purified DNA is ready for library preparation. DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. The ligation reaction was performed by adding 2 μM Illumina's adapters to each sample and incubated at RT one hour. After adapter ligation DNA was PCR amplified with Illumina primers for 16 cycles and library fragments from 180bp to 300bp were isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on Hi-seq3000 following the manufacturer's protocols.
GEO Accession
GSM3677386