Ideal chip seq kit for transcription factors

ChIP was performed with anti-HA-tag antibody (Abcam, ab9110) in MDA-MB-231 PD-L1 KO cells stably expressing HA-tagged PD-L1. For replicate 1, ChIP was performed using Diagenode iDeal ChIP-seq Kit for Transcription Factors (Diagenode, C01010055) with addition of ChIP cross-link Gold cross-lining reagent (C01019027). Library preparation and sequencing analysis were performed at the Molecular Biology Core facility in Dana-Farber Cancer institute. For replicate 2, ChIP was performed as described^{54 (link)}. Qualified libraries were deep sequenced using an Illumina HiSeq 4000 per the manufacturer’s instructions at the Northwestern University.

ChIP was done with rabbit polyclonal anti-FLI1 (C19-X, Santa Cruz Biotechnology) or rabbit IgG control in MHH-ES1 cells using iDeal ChIP-Seq Kit for Transcription Factors (Diagenode). DNA was sheared to an average size of 500 bp to enable mSat2 PCR amplification followed by deep sequencing in an Illumina MiSeq instrument (>42,000X). ChIP efficacy was validated by qRT-PCR using a CCND1 EWSR1-FLI1 binding site^{47 (link)} (positive control) and an intronic CCND1 locus (negative control; Supplementary Fig. 10). Primers are listed in the Supplementary Data.

Chromatin immunoprecipitation (ChIP) was performed using 6 μg of rabbit polyclonal IgG c-Myc antibody (N-262) (sc-764; Santa Cruz Biotechnology, Inc) (undiluted, antibody concentration 200 μg/ml) and the iDeal ChIP-seq kit for Transcription Factors (Diagenode, Denville, NJ, USA) following the manufacturer’s instructions, using CCCTC-binding factor (CTCF) and IgG antibodies (provided with the kit) as positive and negative controls, respectively. The efficiency of ChIP of TERC was expressed as the recovery of the different loci calculated as the percentage of the input (relative amount of immunoprecipitated DNA compared with input DNA), using the formula % recovery = 2^{(Ctinput − Ctsample)}, where Ct_input and Ct_sample are the threshold cycles from the exponential phase of the qPCR for the immunoprecipitated DNA and input, respectively.
Immunoprecipitated DNA was used for qPCR reactions. Primers are listed in the supplementary material, Table S1.

Chromatin immunoprecipitation (ChIP) assay was performed by iDeal ChIP-seq kit for Transcription Factors (Diagenode), according to the manufacturer’s instruction. Briefly, passage 4–6 dermal fibroblasts from patients with dcSSc or healthy controls were fixed with 1% formaldehyde for 10 min. Glycine was added to quench fixation. Cells were lysed and then sonicated on Misonix ultrasonic liquid processor S-4000 for 15 cycles (30 s on/30 s off). MeCP2 antibody, previously used for ChIP-seq in olfactory epithelial tissue^{29 (link)} or rabbit IgG and pre-washed protein A-agarose beads, were mixed and then incubated for 4 hours at 4°C on a rotator. Then 250μL sheared chromatin was incubated with antibody-beads mixed solution at 4°C overnight under constant rotation. Also, 2.5 μL sheared chromatin was put aside as Input. Immunoprecipitated chromatin was eluted from beads using an elution buffer at room temperature with 30 min rotation. Immunoprecipitated chromatin and input were incubated for 4 hours at 65°C to decrosslink chromatin, then DNA was purified using magnetic beads included with the kits. MeCP2 ChIP-seq and input DNA libraries were prepared by the Apollo 324 Next Generation Sample Preparation System with WaferGen reagents, then PCR amplified, cleaned up and sequenced with 50 bp, single-end reads on Illumina Hi-Seq 2500 platform (20 samples pooled together in three lanes).