GSM5203335: RNA-seq_shCTCF_2; Homo sapiens; RNA-Seq
(1) The RNA-Seq assay was performed as previously described. Total RNA product was purified using RNeasy Mini Kit (QIAGEN, Germany) and run-on Agilent Bioanalyzer 4200 to assess RNA integrity. The total RNA‐seq library preparation from 1 μg of total RNA was performed with TruSeq RNA Sample Prep Kit (Illumina, USA) according to the manufacturer's instructions. (2) The ChIP assay was performed as previously described. H3K27ac (ab4729, Abcam, USA), H3K27me3(9733S, CST, USA), CTCF (Millipore, 07-729), H3K9ac (9649, CST, USA), H3K4me (5326, CST, USA), EP300 (ab275378, Abcam, USA) were used for ChIP experiments. For each sample, 100 million crosslinked cells were lysed to prepare nuclear extracts. After chromatin shearing by sonication for 10 min, lysates were incubated on sky wheel overnight at 4°C with protein A Dynabeads (Invitrogen, USA) coupled with 3-6 μg of antibodies. After immunoprecipitation, beads were washed and recovered using magnets. DNA was reverse- cross‐linked at 55°C for 4 hours. After 4 hours, DNA product was purified with QIAquick PCR Purification Kit (QIAGEN, USA). DNA was quantitated using the Qubit dsDNA HS assay and a Qubit 2.0 Fluorimeter (Invitrogen). For ChIP-seq, 5 ng-50ng of purified DNA product were used to generate the DNA library using a VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech, China) and the Illumina HiSeq X Ten was used to sequence the paired-end module and with 150 bp reads on each end (Novogene Biotech, China). For ChIP-qPCR, ChIP products which were used to RT-PCR were performed to amplify the pcr products for 40 cycles using the following PCR conditions: 95 °C for 3 min; and thermocycling at 95°C for 15 s, 60 °C for 60 s, following by 4 °C forever. (3) The ATAC-seq assay was performed as previously described. 1 million fresh cells (THP-1 co-cultured with PANC-1 treated with PMA, TAMs and monocytes) were lysed in 100μL lysis buffer per sample (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) for 10 min on ice to prepare the nuclei briefly. Immediately After 10 min, nuclei were spun for 5 min at 500 x g to remove the supernatant. Then nuclei were incubated at 37 °C with the tagmentation buffer and Tn5 transposome (Vazyme Biotech, China) for 30 min. After the 30 min, ended buffer was added directly into the reaction to quit the tagmentation. Products which were inputted to PCR were performed to amplify the library for 11 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30 s; and thermocycling at 98 °C for 15 s, 60 °C for 30 s and 72 °C for 3 min; following by 72 °C 5 min. After the PCR reaction, DNA libraries were purified with DNA Clean Beads (Vazyme Biotech, China) to remove invalid primer dimmers and quantitate used Qubit 2.0 Fluorimeter (Invitrogen, USA) to quantitate. (4) All eligible DNA libraries were sequenced on the Illumina HiSeq X Ten with 150 bp paired-end reads.