In order to understand what is going on with our DNA/RNA hybrids interactome in our L1 study from EC cells - here is what I want to do.
anti-ORF1p from N2102Ep.
on beads
(1) treat with RNAs A T1 (high salt and low salt)
(2) treat with RNase H
(3) treat with benzonase
(4) BSA control
(5) Additionally - antiORF1p IP, natively elute - S96. IP (with control which will be RNase H treated) - elute using SDS
Save supernatants - but we won’t analyze probably
elute - MS analysis of sensitive fraction
Can we do 3 replicates of each and send
N2102Ep anti-ORF1 IP with Nuclease treatments
Date: 11/17/20
Cell line: N2102Ep
Scale: 50mg N2102Ep with 10ul anti-ORF1 beads
Extraction buffer: 20mM HEPES, pH7.4, 1% Triton x-100, 500mM NaCl (with protease inhibitors)
Weight out 8x 100mg of powder add 200ul extraction buffer (1:4 w:v) per tube; vortex to mix
Sonicate @ 2 Amp for 5x 2sec
Spin @ 20k rcf, 4°C for 10’
Combine clarified lysate from all 8 tubes, evenly splited into 16 tubes
Set up 16x 50mg of anti-ORF1 IPs (3x 5 conditions, one extra for RNase A/T1 high salt followed by native elution with 1mM ORF1 dipeptide (diluted with 20mM HEPEs, 500mM NaCl, no detergent)
IP @ 4°C for 30’
Wash beads 2x 1ml extraction buffer (1st and 2nd washes)
Transfer beads to fresh tubes during 2nd wash
Treat beads with RNase A/T1, RNaseH and Benzonase; use BSA as control
On beads nucleases treatment
RNase A/T1: Themo Scientific, #EN0551, 2mg/ml RNase A, 500U/ml RNase T1 https://www.thermofisher.com/order/catalog/product/EN0551?us&en#/EN0551?us&en
RNAse H: Themo Scientific, # EN0201, 2U/ul https://www.thermofisher.com/order/catalog/product/EN0201#/EN0201
Benzonase: Millipore Sigma, Cat. # 70664, 25U/ul https://www.emdmillipore.com/US/en/product/Benzonase-Nuclease-Purity-990-0,EMD_BIO-70664
Add reaction buffer and enzyme or BSA control to beads (1) treat with RNAs A/T1 (high salt and low salt)
1a_high salt: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (4 replicates)
1b_low salt: 20mM HEPES, pH7.4, 1% Tritonx-100, 100mM NaCl (triplicates)
Add 24ul reaction buffer + 1ul of RNase A/T1
(2) treat with RNase H (triplicates)
Add 2.5ul of 10x RNase H buffer and 21.5ul of nuclease free H20 and 1ul of RNase H
1X Buffer Components: 50 mM Tris-HCl,75 mM KCl, 3 mM MgCl2,10 mM DTT pH 8.3
(3) treat with benzonase (triplicates)
Benzonase reaction buffer: 20mM Tris, pH8, 1% Tritonx-100, 20mM NaCl, 2mM MgCl2
Add 24ul reaction buffer + 1ul Benzonase
(4) BSA control (triplicates)
Add 24ul extraction buffer + 1ul of 2mg/ml BSA
Mix beads with buffer and enzyme completely followed by a brief spin
Incubate @ RT for 30’ with mixing (1000rpm)
Save supernatants after reaction- but we won’t analyze probably
Wash beads 2x 250ul of reaction buffer
Elute beads with 25ul of 2% SDS/40mM Tris, 70C for 5’
Save10% (2.5ul) of each sample, combine three replicates of the same condition, 7.5ul total for Sypro gel (15mg equivalent)
Take one replicate of RNase A/T1 high salt condition, elute with 10ul of 1mM ORF1 dipeptide, RT for 15’ with mixing; load 3 (15mg) and 7ul (35mg) separately on gel
Dry elution in speed vac followed by S-trap protocol and MS analysis (Use new S-trap protocol)
Run 4-12% Bis-Tris, Sypro stain
1) Prestained marker
2) Space
3) RNase A-T1_high_salt_native elution_35mg
4) RNase A-T1_high_salt_native elution_15mg
5) Unstained Marker
6) RNase A-T1_high_salt_SDS_15mg
7) RNase A-T1_low_salt_SDS-15mg
8) RNaseH_SDS_15mg
9) Benzonase_15mg
10) BSA control_15mg
11) BSA_50ng
12) BSA_100ng
Prepare MS sample using new S-trap protocol
1) Solubilize by adding 23μL of 1X lysis buffer to dried sample (or 11.5μL 2X lysis to 11.5μL liquid sample). Mix (sonicate or vortex).
2) Reduce by adding TCEP to 5mM final concentration (1μL of 120mM added to sample) and mix.
a. Incubate 55C for 15 minutes (Thermomixer at 600 RPM).
i. Prepare/label columns during incubation and place in 2mL tubes (lids cut off)
b. Put the tube on ice briefly to bring the temperature down to RT.
3) Alkylate by adding MMTS to 20mM final concentration (1μL of 0.5M MMTS added to sample) and mix.
a. Incubate @ RT for 10 minutes.
b. Prepare protease during incubation
4) Acidify by adding 2.5μL 27.5% phosphoric acid to the sample and mix (final volume 27.5μL)
5) Bind by adding 165μL of S-Trap binding buffer to the sample, pipetting to mix, applying to spin column, and centrifuging 4000g x 30sec (or vacuum). Discard flow-through.
6) Wash protein (try to rotate tubes 180deg between spins if centrifuging, or use vacuum)
a. Wash by adding 150μL S-Trap binding/buffer and centrifuging. (wash 1)
b. Wash x3 by adding 150-200μL 2:1 chloroform:MeOH and centrifuging. (wash 2-4, lipid removal)
c. Wash x3 by adding 150μL S-Trap binding/buffer and centrifuging. (wash 5-7)
7) Digest protein
a. Place S-Trap column into a new Lo-bind tube.
b. Add 20μL of Trypsin/Lys-C solution (125 ng/uL) to the top of the protein trap (2.5μg). Ensure there is no bubble atop the protein trap, can “flick” to help.
c. Cap the S-Trap column loosely and incubate for 1hr at 47C (Thermomixer at 0 RPM with humidification).
8) Elute peptides
a. Apply 40μL 50mM TEAB pH8.5. Centrifuge 4000g x 60sec.
b. Apply 40μL of 0.2% formic acid. Spin.
c. Apply 40μL 50% acetonitrile. Spin.
9) Dry down in Speedvac (~120 minutes at RT)
Check the supernatant of nuclease reaction
Date: 11/23/20
Combine the supernatant after nuclease digestion reactions and BSA control from triplicates of each condition (5 conditions total), load 12.5ul on gel (25mg equivalent). Also load 12.5ul of the supernatant of RNase A/T1 high salt condition which was followed by native elution.
Run 4-12% Bis-Tris, Sypro stain
1) RNase A-T1_high_salt_Sup (same as lane 3; beads were used for native elution)
2) Unstained Marker
3) RNase A-T1_high_salt_Sup
4) RNase A-T1_low_salt_Sup
5) RNaseH_Sup
6) Benzonase_Sup
7) BSA control_Sup
8) BSA_50ng
9) BSA_100ng
10) Space
11) Prestained marker
MS sample preparation
15 elution samples: S-trap micro spin column protocol (modified by LaCava lab)
15 supernatant samples: S-trap ultra-high recovery protocol (see below)
Sample preparation _micro high recovery protocol
Date: 12/09/20
1) Add 23 uL of 5% SDS (final % SDS), 8 M urea, 100 mM glycine pH 7.55 to the (dried down) samples. Combine glycine and urea just before use
This set of samples (supernatant of RNase digestion or BSA control) has no SDS prior to adding S-tray lysis buffer. Add 23ul of 5% SDS, 8 M urea, 100 mM glycine pH 7.55 to each sample.
2) Reduce by adding 1 µL reductant (120 mM aq. TCEP) to sample (5mM final concentration) and mix. 120 mM TCEP: stored @ -20°C. Thaw once and discard the leftover.
3) Incubate 37°C for 15 minutes (Thermomixer at 600 RPM).
4) Put the tube on ice briefly to bring the temperature down to RT. Set Thermomixer to 47°C and humidify.
5) Alkylate by adding 1 uL alkylator (500 mM MMTS). (final concentration: 20 mM)
500 mM MMTS: 63.1mg in 1ml LC-MS isopropanol. Store @ 4°C for 1 month.
6) Incubate @ RT for 15 minutes.
Prepare protease during incubation (see step 10)
7) Acidify by adding 2.5 µL 55% aq. phosphoric acid to the 25 µL sample. This is different than the normal protocol.
55% aq. phosphoric acid: 648ul 85% H3PO4 + 352ul of H2O. Store @ 4°C for months.
8) Place S-trap column into a standard 2.0 mL vial (cut-off lid).
9) Add 165 µL of S-Trap binding buffer (90% MeOH, 100 mM final TEAB, pH 7.55) into the S-Trap micro column.
S-trap buffer: (90% MeOH/100mM TEAB pH 7.55)
10) The next two steps must be done as quickly as possible. Add 2 µg of trypsin/lys-C mix into the acidified sample, immediately mix by pipetting up and down, then immediately transfer the mixture into the S-Trap binding buffer held in the micro spin column. Again mix by pipetting up and down.
Resuspend 20ug of trypsin/lys-C mix in 50ul of resuspension buffer (50mM acetic acid). Use 5ul per sample.
11) Spin in bench-top centrifuge, the S-Trap column in a standard 2.0 mL tube at 4000 g until the solution has passed through (~30 sec). Remove flow through.
12) Wash by adding 150 µL S-Trap buffer (pH=7.55) to the spin column and centrifuging through (4000 g, 30 seconds). Remove flow through. Repeat three times.
Change the direction of the tubes between washes
13) Place S-Trap column into a new 1.7mL Lo-bind tube.
14) Add 0.5 µg of trypsin/lys-C in 25 µL of 50 mM TEAB, pH 8 to the top of the protein trap
10x stock (5ug/25ul): Resuspend 20ug of trypsin/lys-C in 100 µL of 1 mM HCl; dilute to 1x working solution with 55 mM TEAB, pH 8.5 before use (final 50mM TEAB, final pH8.1). If 50mM of acetic acid was used to make 10x stock and diluted to 1x with 55 mM TEAB, pH 8.5, final pH will be 7.7.
15) Cap the S-Trap column loosely and incubate for 2 hrs at 47 °C (with Thermomixer at 0 RPM). DO NOT SHAKE. The cap MUST NOT form an air-tight seal.
16) Add 40 uL of 50 mM TEAB pH 8.5 to the S-Trap column. Centrifuge elution through at 4,000 g for 30 seconds
17) Add 40 uL of 0.2% formic acid. Centrifuge elution through at 4,000 g for 30 seconds
18) Elute hydrophobic peptides with 35 μL 50% acetonitrile, 0.2% formic acid (v:v). Centrifuge elution through at 4,000 g for 30 seconds
19) Dry down in Speedvac (~120 minutes at RT). Store -80 until ready for LC/MS.