Illumina HiSeq 4000 paired end sequencing; Analysis of serum-derived and internalized complement component C3 bound DNA sequences
Analysis of serum-derived and internalized complement component C3 bound DNA sequences
The human lymphoblastoid cell line, Raji was used in our experiments (Raji (ATCC® CCL-86™). The human lypmhoblast Raji cells were cultured in RPMI 1640 medium (Hyclone, SH3025501) supplemented with 10% (vol/vol) Fetal Bovine Serum (Gibco, 10270-016), and penicillin and streptomycin (Hyclone, SV30010). 2 millions of the human lypmhoblast Raji cells/sample were activated or not with 10 μg/mL CpG ODN2006 (Invivogen, #tlrl-2006) overnight at 37oC before incubation with 200 μg/mL C3 in PBS. After treatment, cells were extensively washed in PBS, cross-linked by 1% formaldehyde (Thermo Scientific, SE252317) and sonicated by Bioruptor sonicator (Diagenode) for 25 cycles of 30 s with a 30 s interval (medium intensity) period between cycles, achieving approximately 400-500 bp genomic DNA length after sonication. Lysates were then centrifuged, and the supernatants (sonicated chromatin) were collected. Ten % volume of each sample was removed as the input control. Sonicated chromatin (30 μg) was incubated overnight at 4 °C with 1 μg of polyclonal rabbit anti-human C3c antibody (Dako, #A0062), or a normal rabbit polyclonal IgG (Cell Signaling Technology, #2729) as a negative control. Immune complexes were captured with 10-10 μl mixture of 50% protein A and G coated Dynabeads (Thermo Scientific, 10003D and 10008D) and eluted by reverse cross-linking and protease K digestion for 2 h at 68oC. DNA fragments were purified using ChIP DNA clean and concentrator kit (ZymoResearch, #D0410).  0.5 to 5 ng of ChIPed DNA samples were used to prepare libraries. First, DNA is  end repaired using End-It™ DNA End-Repair Kit (Epicentre, #ER81050) followed by adenylation of 3-end by Klenow Fragment (3'→5' exo) from NEB #M0212L. Later, barcode adapters (Nextflex #228-514174) that contains specific indexes (barcodes) different for each sample so that library from different samples can be mixed together before sequencing. After PCR amplification (18 cycles) using ChIP seq PCR master mix (Nextflex #5143-08, quality assessment of DNA libraries was achieved using Agilent 2100 Bioanalyzer and Quibit quantification. After equimolar pooling of libraries, the final dilution was accurately quantified. At all steps DNA was purified using AMPure XP beads (Beckman coulter #A63881).