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===Experiment/Protocol information=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''for 5X transposome''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | Additional Control|||||||||| |- | Reagent||Stock Conc (uM)||Final rxn conc (uM)||Vol (uL)|||| |- | Glycerol 75%||75%||0.3||14.4||3.6||1.8 |- | Annealed Transposon||50||5||3.6||0.9||0.45 |- | Tn5059||5||2.5||18||4.5||2.25 |- | Total|| || ||36||9||4.5 |- | Total % Glycerol||43.75|||||||| |- | |||||||||| |- | |||||||||| |- | incubate at RT, 30m|||||||||| |- | store -20C|||||||||| |- | |} 1. info *resuspend 1 tablet roche protease inhibitor in 1.5 mL N-H2O. Next day add 8.5 mL, then take 920 uL and add to 80 uL 50X LB Repeater combitips 100 uL combitip at 1 uL = 2.5 uL 100 uL combitip at 7 uL = 8.5 uL 200 uL combitip at 10 uL = 12.0 uL 200 uL combitip at 12 uL = 14.0 uL 500 uL combitip at 15 uL = 20.0 uL 1000 uL combitip at 150 uL = 157.0 uL *Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O. *FACS buffer: 2X (PBS, 4 mM EDTA, 2% BSA) (2X = 240 mg BSA, 96 uL 500 mM EDTA, 11.6 mL PBS) (1X = 3 mL 2X, 3 ml NH20) *2. Samples samples before FACS sorting 1-96. scT7_r0XX_i5, 5X tn5059, ~2,000 nuclei per rxn, buffer D6, samples 1-96 after FACS sorting 1-92. 25 nuclei, 1% BSA FACS buffer, gHCl after sort, pooled 1-96 samples 93. 25 nuclei positive IVT control 12E 94. 25 nuclei positive IVT control 12F 95. NTC control 12G 96. NTC control 12H
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