title: GSM5311517: HMGA2-OE MKN-45- input; Homo sapiens; ChIP-Seq
library_construction_protocol: HMGA2-OE MKN-45 cells (4x106 cells per ChIP) were protein-DNA cross-linked with 1% formaldehyde at 37°C for 10 minutes and then the reactions were ceased with glycine (125mM). The cells were lysed and the chromatins were enzymatic digested with Micrococcal Nuclease (0.0125mL/mL) at 37°C for 20min. The nuclear membranes were disrupted using a Uitrasonic Homogenizer (JY92-IIN) for 10min. The size and the concentration of the purified DNA from the digested chromatins were measured for the quality control via electrophoresis and NanoDrop 2000, respectively. The digested chromatins were then performed the immunoprecipitation with the HMGA2 rabbit mAb (CST, #5269S). Histone H3 (D2B12) XP® Rabbit mAb (CST, #4620) acted as the positive control, and Normal Rabbit IgG (CST, #2729) acted as the negative control correspondingly. After immunoprecipitation, the precipitated protein-DNA were de-crosslinked, purified and checked by qPCR to verify the results of the immunoprecipitation, and the primer we used was SimpleChIP® Human RPL30 Exon 3 Primers 1(CST, #7014). IP efficiency was calculated with the Percent Input Method. SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads, CST, #9005) was used in the experiments. The DNA was quality evaluated through NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and Qubit® DNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). After the DNA was convinced qualified, the cDNA library was prepared and sequenced on Illumina's Novaseq platform to generate 150 bases pair-end reads.