title: Illumina HiSeq 2000 paired end sequencing; ChIP-seq of H3K4me3 and H3K27ac of dermal fibroblasts
design: ChIP-seq of H3K4me3 and H3K27ac of dermal fibroblasts
library_name: HS3_H3K4me3_PIC4_p
library_construction_protocol: Following stimulation, sample were crosslinked in 1% HCHO (prepared in 1X DPBS) at room temperature for 10 minutes, and HCHO was quenched by the addition of glycine at a final concentration of 0.125M. Cells were pelleted at 4°C at 2000 x g, washed with ice-cold 1X DPBS twice, and snapped frozen in liquid nitrogen. The cell pellets were stored in -80°C until further stages were performed.  Human dermal fibroblasts were obtained from the HipSci consortium (http://www.hipsci.org/ (Streeter et al., 2017) (where they were used for creating iPSCs). These original cells were thawed to test for growth and viability and refrozen after 1-2 passages, so that all lines were in the same growth rate and proliferation conditions before the stimulation experiment. Non-human primate cells were extracted from skin tissues that were incubated for 2hrs with 0.5% collagense-A (Roche; 11088815001) after mechanical processing, and then filtered through 100µm strainers before being plated and passaged prior to cryo-banking. Rodent cells were obtained from Cellbiologics where they were extracted using a similar enzymatic extraction protocol using skin tissues from the same regions of the body used in primates. Cell were grown in ATCC fibroblast growth medium (Fibroblast Basal Medium (ATCC, ATCC-PCS-201-030) with Fibroblast Growth Kit-Low serum (ATCC, PCS-201-041)  Cells were stimulated with 1ug/mL High-Molecular Weight poly I:C (Invivogen, Cat. Code: tlrl-pic) transfected with 2uL/mL Lipofectamin 2,000 (ThermoFisher, Cat Number 11668027), for 4 hours. ChIPmentation was performed according to version 1.0 of the published protocol (Schmidl et al., 2015) with some modifications at the ChIP stage: Cell pellets were lysed in 300 ul ChIP Lysis Buffer I (50mM HEPES.KOH, pH 7.5, 140mM NaCl, 1mM EDTA, pH 8.0, 10% Glycerol, 0.25% NP-40, 0.5% Triton X-100) on ice for 10 minutes. Cells were then pelleted at 4°C at 2000 x g for 5 minutes, and washed by 300ul ChIP Lysis Buffer II (10 mM Tris.Cl, pH 8.0, 200 mM NaCl, 1mM EDTA, pH 8.0, 0.5mM EGTA, pH 8.0), and pelleted again at 4°C at 2000 x g for 5 minutes. Nuclei were resuspended in 300 ul ChIP Lysis Buffer III (10mM Tris.Cl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-Lauryolsarcosine). Chromatin was sonicated using Bioruptor Pico (Diagenode) with 10 cycles of 30 seconds ON/30 seconds OFF. 30ul 10% Triton X-100 were added into each sonicated chromatin, and insoluble chromatin was pelleted at 16,100 x g at 4°C for 10 minutes. 1ul supernatant was taken as an input control. The rest of the supernatant was incubated with 10ul Protein A Dynabeads (Invitrogen) pre-bound with 1ug anti-H3K4me3 (Diagenode Cat Number C15410003) or 1ug anti-H3K27ac (Diagenode, Cat Number C15410196) in a rotating platform in a cold room overnight. Each immunoprecipitation (IP) was washed with 500ul RIPA Buffer (50mM HEPES.KOH, pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Sodium Deoxycholate, check components) for 3 times. Each IP was then washed with 500ul 10mM Tris, pH 8.0 twice, and resuspended in 30ul tagmentation reaction mix (10mM Tris.Cl, pH 8.0, 5mM Mg2Cl, 1ul Tn5 transposase (Nextera)). The tagmentation reaction was put on a thermomixer at 37°C for 10 minutes at 800 rpm shaking. Following tagmentation, each IP was washed sequentially with 500ul RIPA Buffer twice, and 1X TE NaCl (10mM Tris.Cl, pH 8.0, 1mM EDTA, pH 8.0, 50mM NaCl) once. Elution and reverse-crosslinking were done by resuspending the beads with 100ul ChIP Elution Buffer (50mM Tris.Cl, pH 8.0, 10mM EDTA, pH 8.0, 1% SDS) on a thermomixer at 65°C overnight, 1,400 rpm. DNA was purified by MinElute PCR Purification Kit (QIAGEN, Cat Number 28004) and eluted in 12.5ul Buffer EB (QIAGEN, Cat Number 28004), which yielded ~10ul ChIPed DNA. The library preparation reactions contained the following reagents: 10ul purified DNA (from the above procedure), 2.5ul PCR Primer Cocktails (Nextera kit, Illumina, Cat Number FC-121-1030), 2.5ul N5xx (Nextera index kit, Illumina Cat Number FC-121-1012), 2.5ul N7xx (Nextera index kit , Illumina, Cat Number FC-121-1012), 7.5ul NPM PCR Master Mix (Nextera kit, Illumina, Cat Number FC-121-1030) The PCR cycles were as follows: 72°C, 5 mins; 98°C, 2 mins; 98°C, 10 secs, 63°C, 30 secs, 72°C, 20 secs] X 12; 10°C hold. The amplified libraries were purified by double AmpureXP beads purification: first with 0.5X bead ratio, keep supernatant, second with 1.4X bead ratio, keep bound DNA. Elution was done in 20ul Buffer EB (QIAGEN).
attributes.tag: Experimental Factor: organism
attributes.value: Homo sapiens
attributes.tag: Experimental Factor: stimulus
attributes.value: poly I:C
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: H3K4me3