title: Illumina HiSeq 2500 paired end sequencing; Effect of LINC00899 depletion on histone mark enrichment in HeLa cells
design: Effect of LINC00899 depletion on histone mark enrichment in HeLa cells
library_construction_protocol: Cells were washed once with phosphate buffered saline solution, spun down at 600g for 3 minutes and washed twice with 1.5 mL Wash buffer (20 mM HEPES-KOH at pH 8.5, 150 mM NaCl, 0.5 mM spermidine and 1X cOmplete EDTA-free protease inhibitor cocktail). HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 degrees Celsius in 5% CO2. For RNA interference, cells were transfected with either the negative control siRNA or a siRNA targeting LINC00899. For antisense oligonucleotides (LNAs), cells were transfected with either the negative control LNA A or a LNA targeting LINC00899 (LNA #2). All transfections were performed with RNAmax in 30-mm dishes, and cells were left in culture for 48 hours after transfection. Concanavalin A-coated magnetic beads were washed twice in 1.5 mL binding buffer (20 mM HEPES-KOH at pH .5, 10 mM KCl, 1 mM CaCl2, 1M MnCl2) and resuspended in 10 uL binding buffer. Cells were mixed with beads, rotated for 10 minutes at room temperature, and collected by binding to the magnetic beads. Cells were resuspended in 50 uL antibody buffer (wash buffer with 0.05% digitonin and 2 mM EDTA) with antibodies in 1:100 dilution. Antibodies were used against H3K4me3 (Millipore 05-1339 CMA304, Lot2780484), H3K27me3 (Cell Signaling #9733S C36B11, Lot8), H3K36me3 (Active Motif Cat#61101, Lot 32412003) and H3K27ac (Abcam ab4729, Lot GR3211741-1), as well as a goat anti-rabbit IgG H&L (Abcam ab97047, Lot GR254157-8). Cells were incubated with the antibodies overnight at 4 degrees Celsius, and then washed once with 1mL Digitonin buffer (Wash buffer with 0.05% Digitonin). For the mouse anti-H3K4me3 antibody, samples were incubated with 50 ul of a 1:100 dilution in Digitonin buffer of secondary rabbit anti-mouse antibody for 10 minutes at RT and then washed once with 1 mL Digitonin buffer. Samples were then incubated in 50 ul Digitonin buffer containing 700 ng/ml Protein A-MNase fusion protein for 10 minutes at room temperature followed by two washes with 1mL Digitonin buffer. Cells were then resuspended in 100ul Digitonin buffer and cooled down to 4C before addition of CaCl2 to a final concentration of 2mM. Targeted digestion was performed for 30 minutes on ice until 100ul of 2X STOP buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.02% Digitonin, 250mg RNase A, 250ug Glycogen, 15pg/ml yeast spike-in DNA) was added. Cells were then incubated at 37 degrees Celsius for 10 minutes to release cleaved chromatin fragments, spun down for 5 minutes at 16,000g at 4 degrees Celsius and collected on magnet. Supernatant containing the cleaved chromatin fragments were then transferred and cleaned up using the Zymo Clean & Concentrator Kit. Library preparation was performed using the ThruPLEX DNA-Seq library preparation kit from Rubicon Genomics. A modified library amplification programme was used, consisting of extension for 3 minutes at 72 degrees Celsius; cleavage for 2 minutes at 85 degrees Celsius; denaturation for 2 minutes at 98 degrees Celsius; addition of indices by four cycles of 20 seconds at 98 degrees Celsius, 20 seconds at 67 degrees Celsius and 40 seconds at 72 degrees Celsius; and amplification for 12 cycles of 20 seconds at 98 degrees Celsius and 15 seconds at 72 degrees Celsius (14 cycles were used for DNA pulled down with the goat anti-rabbit antibody). The average library size was tested using an Agilent Tapestation DNA1000 High Sensitivity Screentape and quantification was performed using the KAPA library quantification kit.
attributes.tag: Experimental Factor: loss of function method
attributes.value: LNA
attributes.tag: Experimental Factor: compound
attributes.value: negative control
attributes.tag: Experimental Factor: RNA interference
attributes.value: not applicable
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-H3K36me3 (Active Motif)