title: GSM4601560: GC0713477_S194; Homo sapiens; OTHER
library_construction_protocol: The fixed cells were thawed on ice. To prepare PA-MNase and antibody complex, 1 μl antibody and 3 μl PA-MNase were pre-incubated on ice in 4 μl antibody binding buffer (10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 150 mM sodium chloride, 0.1% Triton X-100) for 30 min. Meanwhile, H1 fixed cells (1 million) and HEK 293T fixed cells (1 million) were resuspended in 100 μl antibody binding buffer. Then, cell suspension was added to the PA-MNase and antibody complex, incubated on ice for 1 hour. Cells were washed three times with high salt buffer (10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 400 mM sodium chloride and 1% (v/v) Triton X-100), followed by washing once with rinsing buffer (10 mM Tris pH7.5, 10 mM sodium chloride and 0.1% (v/v) Triton X-100). Then the cells were resuspended in 40 μl reaction solution buffer (10 mM Tris-Cl (pH 7.4), 10 mM sodium chloride, 0.1% (v/v) Triton X-100, 2 mM CaCl2), incubated at 37°C for 3min in water bath. The reaction was stopped by adding 4.4 μl 100mM EGTA. After washing twice with rinsing buffer, the cells were end-repaired by T4 Polynucleotide Kinase (PNK) in 150 μl reaction buffer (1 x PNK buffer, 1mM ATP, 150 unites PNK) at 37°C for 30min, followed by washing twice with rinsing buffer to stop the reaction.The cells were resuspended in 25 μl reverse transcription buffer (5 μl 10 x Maxima H Minus reverse transcription buffer, 1.25 μl 10% NP40, 16.75 μl H2O, 1 μl 100 μm not-so-random primer, 1 μl 10ng/μl Oligo dT22 primer (NNNNNNGAGCGTTTTTTTTTTTTTTTTTTTTTTVN)). After incubated at 65°C for 1min, the reaction was immediately put on ice, while the enzyme mix is prepared (8.75 μl H2O, 5 μl 10 x Maxima H Minus reverse transcription buffer, 8 μl 10 mM dNTPs, 2 μl Maxima H Minus reverse transcriptase, 0.625 μl SUPERase• In™ RNase Inhibitor, 0.625 μl RNaseOUT™ Recombinant Ribonuclease Inhibitor) and added into the reaction. The reverse transcription is performed as described in [15] (50 °C × 10 min; 3 cycles for the following: 8°C × 12s, 15°C × 45s, 20°C × 45s, 30°C × 30s, 42°C × 2min, 50°C × 5 min; 50 °C × 10 min and hold at 4 °C). The cells were washed twice with rinsing buffer, resuspended in 50 μl reaction buffer (5 μl 10 × Exo I buffer, 1 μl Exo I, 44 μl H2O) and incubated at 37°C for 20min. This is to remove the excess primers left after reverse transcription. After digestion, the cells were washed twice with rinsing buffer to stop the reaction. 96 barcode-P7 adaptors (10 μM) stored in a 96 well plate were thawed at 4 °C, then 1 μl of each was added to the corresponding well in a new 96 well plate with multichannel pipette. Downstream library construction was performed as described previously. Briefly, the cells were suspended with nuclei suspension buffer and mixed with enzyme dilution buffer, followed by aliquoted into 10 μl in 96 wells, mixing with the added barcode-P7 adaptors. The plate was sealed completely and incubated at 37°C for 60min. After incubation, the cells were pooled together in a solution trough containing 500 μl stop buffer, resuspended with 800 μl 1× PBS and send to flow cytometry core. 30 cells were sorted in each well of a new 96 well plate which contain 13 μl buffer mixture per well (3 μl reverse-crosslink buffer, 10 μl PBS containing 0.1% NP40). The plate was sealed completely and incubated at 65°C for 6 hours and 80°C for 10 min. After reverse crosslink, indexed PCR1 was performed by adding 13 μl 2x Phusion® High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs) and 1 μl 2 μM index primer with the following condition: 98 °C 3 min, 12 cycles of 65 °C 30s, 72 °C 30s, followed by 72 °C 5 min. Then the libraries were pooled together, digested with Exo I and purified by MinElute® Reaction Cleanup Kit (Qiagen). Downstream A-tailing and P5 adaptor ligation were performed as described previously. PCR2 amplification with i5 index primer and P7-cs2 primer was set in the following condition:  98 °C 3 min, 57 °C 3 min, 72 °C 1 min, 7 cycles of 98 °C 10s, 65 °C 15s, 72 °C 30s, followed by 72 °C 5 min. The PCR products were run on the 2% E-Gel® EX Agarose Gel (Invitrogen). The fragments between 250–600 base pair (bp) were isolated and purified by the MinElute Gel Extraction Kit (Qiagen). The concentration of the library was measured by Qubit dsDNA HS kit (Thermo Fisher Scientific). The paired-end sequencing was performed on Illumina Hiseq 2500
attributes.tag: GEO Accession
attributes.value: GSM4601560