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Alice:Whole Genome Bisulfite Sequencing Lab Notes/WGBS
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===Validation of DMRs identified=== *validate some DMRs identified above in all other mSCNT, miPSC lines *samples to be processed for bisulfite conversion: **SCNT-ESC (official ID): NB4 (SCNT4), B7 (SCNT5), B8 (SCNT6), B9 (SCNT7), B10 (SCNT8), B11 (SCNT9), B13 (SCNT11) **iPSC (official ID): miPS1 (miPSD1), miPS3 (miPSD3), miPS4 (miPSD8), 2eiPS2 (2eiPS2P5) **MEF(official ID): B6P1-1(), B6P1-2(), TB_MEF(), ZN_MEF() *use 500ng of gDNA as input '''Bisulfite conversion''' *use EZ-96 DNA Methylation-Lightning™ MagPrep kit and protocol **Add 130 µl of Lightning Conversion Reagent to 20 µl of a DNA sample in a Conversion Plate. Mix the samples by pipetting up and down. **Seal the plate with the provided film. Transfer the Conversion Plate to a thermal cycler and perform the following steps: #98°C for 8 minutes #54°C for 60 minutes (then 4°C storage for up to 20 hours if needed) **Pre-heat a plate heating element to 55°C. **Add 600 µl of M-Binding Buffer and 10 µl of MagBinding Beads to each well of a Collection Plate. **Transfer the samples from the Conversion Plate into the Collection Plate containing the M-Binding Buffer and MagBinding Beads. Mix by pipetting up and down 3-6 times and/or vortexing at 1,300-1,500 rpm for 30 seconds **Let plate stand at room temperature for 5 minutes, then transfer plate to a magnetic stand for an additional 5 minutes or until beads pellet and supernatant is cleared. With the plate on the magnetic stand remove the supernatant and discard. **Remove the Collection Plate from the magnetic stand for this and each subsequent buffer addition. Add 400 µl of M-Wash Buffer to the beads. Re-suspend the beads by pipetting up and down or vortexing the plate at 1,300-1,500 rpm for 30 seconds. Replace the plate on the magnetic stand for 3 minutes or until beads pellet. Remove and discard supernatant. **Add 200 µl of L-Desulphonation Buffer to the beads. Re-suspend the beads by pipetting up and down or vortexing for 30 seconds. Let plate stand at room temperature (20-30°C) for 15-20 minutes. After the incubation, replace the plate on the magnetic stand for 3 minutes or until beads pellet. Remove and discard supernatant. **Add 400 µl of M-Wash Buffer to the beads. Re-suspend the beads by pipetting up and down or vortexing for 30 seconds. Replace the plate on the magnetic stand for 3 minutes or until beads pellet. Remove and discard supernatant. Repeat this wash step. **Transfer the plate to a heating element at 55°C for ~15 minutes to dry the beads (be careful don't over-dry) and remove residual M-Wash Buffer. **Add 30 µl of M-Elution Buffer directly to the dried beads and pipette or vortex for 30 seconds to re-suspend. Heat the elution at 55°C for 4 minutes then transfer the plate to the magnetic stand for 1 minute or until beads pellet. Remove the supernatant and transfer to a clean Elution Plate. **use ~10ng of converted DNA for subsequent PCR '''PCR''' *q-PCR to check the bisulfite converted DNA *Perform in 50ul reaction with 1ul of bis-cvt DNA {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''volume (ul)''' |- | bis-cvt DNA||1.00 |- | 10uM Me-Enhancer primer F||1.00 |- | 10uM Me-Enhancer primer R||1.00 |- | 2x KAPA fast MM ||25.00 |- | H2O||21.00 |- | Total||50.00 |} '''Program''' *98C 3min --> (98C 10sec -> 60C 30sec -> 72C 45sec) x 15 cycles --> 72C 3min --> 15C Hold *The PCR didn't work, so I quickly stopped and changed annealing temperature *98C 3min --> (98C 10sec -> 58C 30sec -> 72C 45sec) x 12 cycles --> 72C 3min --> 15C Hold *PAGE gel results: ====Sequencing library preparation==== *In order to be consistent with WGBS data, I will use IQ MM for PCR instead of KAPA *The primer design of first round and second round PCR can be found here: **briefly, we first design primers that span part (or entire) region of the DMR identified. Then we add part of the Y-adaptor onto the end of both primers. **It is the complementary part of the Y-adaptor that will be added onto the end of both primers, then we can use PCR_F and PCR_N2R for 2nd round PCR **add in front of all Forward primer: 5' GCGAGAAGGCTAG 3' **add in front of all Reverse primer: 5' GCGAGAAGGCTAG 3' *sample info: {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''sample name''' | align="center" style="background:#f0f0f0;"|'''N2 barcode''' |- | 1||NB1||PCR_N2R10 |- | 2||SCNTB7||PCR_N2R11 |- | 3||SCNTB8||PCR_N2R12 |- | 4||SCNTB9||PCR_N2R13 |- | 5||SCNTB10||PCR_N2R14 |- | 6||SCNTB11||PCR_N2R15 |- | 7||SCNTB13||PCR_N2R16 |- | 8||B6P1-1||PCR_N2R17 |- | 9||B6P1-2||PCR_N2R18 |- | 10||miPSD1||PCR_N2R19 |- | 11||miPSD3||PCR_N2R20 |- | 12||miPSD8||PCR_N2R21 |- | 13||ZN_MEF||PCR_N2R22 |- | 14||TB_MEF||PCR_N2R23 |- | 15||2eiPS2P5||PCR_N2R24 |} '''PCR''' {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''9 rxn mix (9 primer pairs)''' |- | bis-cvt DNA||1.00||0.00 |- | 10uM Me-Enhancer primer F||1.00||0.00 |- | 10uM Me-Enhancer primer R||1.00||0.00 |- | 2x iQ MM (Biorad)||25.00||188 |- | H2O||21.00||160 |- | Total||50.00||375 |} '''PCR program''' *after two failed runs with 98C as denaturing temperature, final program used for big batch processing: 95C 2min --> (95C 15sec -> 58C 20sec -> 72C 30sec) x 26-28 cycles --> 72C 3min --> 15C Hold *combine the PCR amplicon of different targets into the same tube for the same sample, then perform Qiaquick column purification and elute in 33ul H2O *so now there are 15 samples (each with all 9 targets combined into one tube) to run 2nd PCR '''2nd PCR with Illumina primers''' {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''15 rxn mix''' |- | bis-cvt DNA targets pooled||10.00||- |- | 10uM PCR F||1.00||16.00 |- | 10uM PCR R_N2 barcoded||1.00||- |- | 2x iQ MM (Biorad)||25.00||380 |- | H2O||21.00||315 |- | Total||60.00|| |} '''PCR program''' 95C 2min --> (95C 15sec -> 60C 30sec -> 72C 30sec) x 32 cycles --> 72C 3min --> 15C Hold *Qiaquick column purification and elute in 42ul H2O each (15 total) *loaded 2ul of purified samples for PAGE gel validation *PAGE gel results: samples 1,2,4,9,13 had high yield, while the rest of them had very concentrations *so I did the pooling based on eye-balling the gel results: **3ul of the samples with high yield (sample 1, 2, 4, 9, 13) **12ul of the samples with relative low yield (sample 10, 11, 14) **15ul of the samples with low yield (sample 3, 5, 6, 7, 8, 12, 15) *after pooling, used the total volume ~156ul for gel size selection (~120-220bp bands) *ethanol precipitation and elute in total 60ul H2O (blue color due to ~3ul of glycoblue per tube, total of 18ul, was added during first step) *gel validation *Results: the sequences seem to be over 90% junk reads that mapped to other organisms. **Maybe due to difficulty to amplify non-coding region, and the primers include too many mismatches because of the Illumina primers attached to the end.
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