GSM4984362: USA300_WT_biolrep2_techrep1; Staphylococcus aureus subsp. aureus USA300; OTHER
For the CRAC experiments, the cells were washed off the filters with 25ml of ice-cold Phosphate Buffer Saline (PBS) and pelleted by centrifugation. Cells (up to 0.5 grams) were subsequently resuspended in 2 volumes/cel weight of TN150-Lysostaphin buffer (50 mM Tris pH 7.8, 150 mM NaCl, 100 µg/mL Lysostaphin, 0.1% NP-40, 0.5% Triton X-100) and transferred to 5 mL screw-cap tubes (eppendofr). Sixty µL of RQ DNase 1 and 10 µL of SUPERase·In was added to the mixture and incubated at 37°C for 10 min to lyse the cells. 3V/cell weight of Zirconia beads (Thistle Scientific; 0.1 µm) were added and the mixture was vortexed vigorously 5 times for one minute with one-minute incubations on ice between each step. 2V/cell weight of cold TN150 anti-peptidase (50 mM Tris pH 7.8, 150 mM NaCl, 1 Roche EDTA-free mini pellet, 0.1% NP-40, 0.5% Triton X-100 and 10 mM EDTA) buffer before centrifugation for 30 minutes at 10000 g at 4°C. Subsequently, 75µl Anti-FLAG® M2 Magnetic Beads (pre-washed with 3 times 1 mL TN150 buffer) was added and incubated with the lysate for 2 hours at 4ºC. The beads were then washed three times five minutes with 2 mL TN1000 buffer (50 mM Tris pH 7.8, 1M NaCl, 0.1% NP-40, 0.5% Triton X-100) and three times with 2 mL TN150 buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% NP-40, 0.5% Triton X-100) for 5 minutes. Beads were subsequently resuspended in 250 µl of TN15, 10 µL of home-made GST-TEV protease was added and the samples were rotated for 1.5-2 hours at room temperature. TN150 was added to a final volume of 600 µL and 550 µL of the TEV eluate was incubated with RNace-it™ (Agilent Technologies1 µL of 1:100 dilution) for exactly five minutes at 37ºC. Subsequently, 500 µl of the mixture was added to a tube with 0.4 g Guanidium-HCl and vortexed vigorously to inactivate the RNases. NaCl and imidazole (pH 8.0) were added to a final concentration of 300 mM and 10 mM, respectively and the mixture was transferred to 50µl of Ni-NTA agarose beads (QIAGEN) equilibrated with Wash buffer I (300 mM NaCl, 10 mM imidazole, 6M GuHCl, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM β- mercaptoethanol (βMe) and 0.5% Triton X-100) and incubated at 4ºC overnight on a rotator. The next day the beads were transferred to Pierce snap-cap columns (Thermo Scientific) and washed twice with 500 µL Wash buffer I and three times with 500 µL 1x NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM βMe and 0.5% Triton X-100). Radioactive labelling of 5' ends, linker ligation and cDNA library preparation was performed as described previously (McKellar et al., 2020).