GSM3560256: H1_IgG_(20180919_HK_Hs_H1_IgG_0912); Homo sapiens; OTHER
High-throughput CUT&Tag: Cells were washed twice with Wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1X Protease inhibitor cocktail) and resuspended in Dig-wash (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1X Protease inhibitor cocktail; 0.05% Digitonin) containing 2mM EDTA and arrayed in a 96-well plate, 25 µL per well. Permeabilization before antibody incubation varied from 1 to 5 hours. Then, 25 µL 1:25 dilutions of appropriate antibodies in Dig-wash-EDTA were added. Cells were incubated with primary antibodies overnight at 4 oC. Concanavalin A (ConA) coated magnetic beads (Bangs Laboratories) were prepared as described [Skene, P.J. & Henikoff, S. Elife 6:e21856 (2017)]. Ten µL of activated ConA-coated magnetic beads were added to each sample, mixed gently and incubated at room temperature for 10 minutes. The plate was placed on a magnetic plate holder and supernatants were discarded. An appropriate secondary antibody (Guinea Pig anti-Rabbit IgG Heavy & Light Chain antibody, Antibodies-Online ABIN101961) was prepared as a 1:50 dilution in Dig-wash and added to each well. Cells were washed 3 times with Dig-wash and then incubated with a 1:200 dilution of pA-Tn5 adapter complex (Kaya-Okur et al., CUT&Tag for efficient epigenomic profiling of small samples and single cells, manuscript submitted) in Dig-med (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at RT for 1 hr. Cells were washed 3x for 5 min in Dig-med, resuspended in 50 µL Tagmentation buffer (Dig-med + 10 mM MgCl2) and incubated at 37 for 1h. To stop tagmentation, 2.25 µL 0.5M EDTA, 2.75 µL 10% SDS and 0.5 µL 20 mg/ml Proteinase K was added to the sample, which was incubated at 55 oC for 30 min and then at 70 oC for 20 min to inactivate the Proteinase K. Samples were held at 4oC overnight until ready to continue. A 1.1x volume of AMPure XP beads was added to each well, vortexed and incubated at room temperature for 10-15 min. The plate was placed on magnet and unbound liquid was removed. Beads were gently rinsed twice with 80% ethanol, and DNA was eluted with 35 µL of 10 mM Tris-HCl pH 8. A 21 µL sample was mixed with 2 μL of a universal i5 and a uniquely barcoded i7 primer [Buenostro, J.D. et al. Nature 523, 486-U264 (2015)], using a different barcode for each sample. A volume of 25 μL NEBNext HiFi 2x PCR Master mix was added and mixed. The sample was placed in a Thermocycler with heated lid using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 sec; 14 cycles of 98 °C for 10 sec and 63 °C for 30 sec; final extension at 72°C for 1 min and hold at 8 oC. Post-PCR clean-up was performed by adding a 1.1X volume of Ampure XP beads (Beckman Counter), and libraries were incubated with the beads for 15 min at RT, washed twice gently in 80% ethanol, and eluted in 30 μL 10 mM Tris pH 8.0. All 96 individually i7-barcoded libraries were mixed in equal proportions for sequencing on an Illumina HiSeq 2500 2-lane (Turbo) flow-cell. High-throughput CUT&Tag
GEO Accession
GSM3560256