AlanFung:LabNotes/CTCF/2011-5-16: Difference between revisions
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===2nd Strand Synthesis and Library Enrichment=== | ===2nd Strand Synthesis and Library Enrichment=== | ||
*Mix on ice | *Mix on ice | ||
0.5 uL Short Orange | 0.5 uL Short Orange | ||
0.5 uL Short Blue | 0.5 uL Short Blue | ||
10 uL DNA/Water | 10 uL DNA/Water | ||
1 uL Klenow | 1 uL Klenow | ||
25 uL KAPA Supermix | 25 uL KAPA Supermix | ||
13 uL Water | 13 uL Water | ||
50ul total | 50ul total |
Revision as of 17:22, 17 May 2011
Nextera Methyl-Seq DNA Sample Prep on 50ng 100pg & 600pg DNA with negative control
Low input transposase-based library preparation (Shendure's Protocol)
Objective
- Compare 100pg starting material prepared by column purification to bead bind purification
- Confirmation of the library prep with beta nextera methyl-seq kit with input DNA (100 cells, ~600pg DNA starting amount) with GM12878 gDNA.
- Adapt Shendure's protocol for low input library preparation
- Increase yield from the bisulfite conversion using zymo methylation direct kit (Claim to work with 50pg starting amount)
- Improve the yield from the bisulfite conversion step using suggestions made by zymo tech support
- Perform fill-in with mC by Klenow DNA polymerase prepared by Arthurva since we used up the one from Nextera Methyl Seq beta one
- Perform 2nd strand synthesis after bisulfit conversion with Klenow which helps the protecting the fragile DNA structure after bisulfite conversion
- Include carrier in column purification during bisulfite conversion, in order to minimise DNA lost
Samples & Materials
- Nextera Sample Prep Kit_Epicentre_NMS072910B
NexteraTM Methyl-Seq Tagmentation Mix (Illumina-compatible) 5X Nextera Reaction Buffer (HMW) 50X Nextera Primer Cocktail (Illumina-compatible) 50X Nextera Adaptor 2 (Illumina-compatible) 2X Nextera Fill-in Buffer 2X Nextera PCR Buffer 200X Nextera Read 1 Primer 200X Nextera Read 2 Primer 200X Nextera Index Read Primer Nextera Methyl-Seq Polymerase
- Input DNA
- Bisulfite conversion kit (Methylation Direct Kit)
Overview
- Tagmentation with diluted transposon at low volume (5ul)
- Fill-in with Klenow (DNTP-mC)
- Bisulfite Conversion with Zymo EZ DNA methylation Direct Kit
- Column Purification with carrier
- 2nd strand synthesis with short orange/blue primer using Klenow DNA polymerase
- Library enrichment with orange/blue primer using KAPA
Protocol
TagmentationTM Reaction
- Dilute GM12878 gDNA 1:100 to 176ng/ul
- 1. Prior to assembling the reaction, briefly centrifuge ALL tubes to assure that the reagents are at the bottom of the tubes.
- 2. Assemble the following reaction components on ice, in the order listed:
Tube A 0.568ul 100pg NA12878 gDNA (176pg/ul qubit check on 03/31/11) 2.432ul ddh2O 1ul 5X Nextera HMW Reaction Buffer 1ul 1:50 Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible) ((1 μl Nextera enzyme, 24 μl TE, 25 μl 100% gly- cerol) 5ul Total reaction Volume
Tube B 3.41ul 600pg NA12878 gDNA (176pg/ul qubit check on 03/31/11) 1ul 5X Nextera HMW Reaction Buffer 1ul 1:50 Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible) ((1 μl Nextera enzyme, 24 μl TE, 25 μl 100% gly- cerol) 5.4ul Total reaction Volume
Tube C 3ul ddh2O 1ul 5X Nextera HMW Reaction Buffer 1ul 1:50 Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible) ((1 μl Nextera enzyme, 24 μl TE, 25 μl 100% gly- cerol) 5ul Total reaction Volume
Tube D 1ul 50ng NA12878 gDNA (50ng/ul) 2ul ddh2o 1ul 5X Nextera HMW Reaction Buffer 1ul 1:50 Nextera Methyl-Seq Tagmentation Mix (Illumina-compatible) ((1 μl Nextera enzyme, 24 μl TE, 25 μl 100% gly- cerol) 5ul Total reaction Volume
- 3. Mix briefly by pipetting, and incubate at 55C for 5 minutes.
- Add 1 uL Klenow Polymerase and 1 uL methyl-Biotin-dNTP Mix
- Incubate for 40min at 30 C
Bisulfite Conversion of Tagged Nextera Fragments
- Add 13ul ddh2o to all tubes make it up to 20ul
- Add in 130uL of CT conversion Reagent Solution directly to the digested samples
- Vortex and spin down
- Perform reaction in thermocycler
Step1 98C, 8m Step2 64C, 3.5hr Step4 4C, storage for up to 20 hr
- Add 600uL of M-Bindin Buffer into a IC Column
- Load 150uL of samples into IC column
CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES
- Centrifuge at 14,000rpm for 30s
- Discard flow through
- Add 200uL of M-Desulphonation Buffer to column let stand at RT for 14m
- Repeat centrifuge step
- Add 200uL of M-Wash Buffer to the column
- Repeat Centrifuge
- Place column in a 1.5mL tube
- Add in 11uL of warm M-Elution Buffer (37C) directly to the column matix, sit at 37C for 5 min
- Repeat Centrifuge
Dilution of 50ng positive control
- ssDNA qubit quantification
- Results
2nd Strand Synthesis and Library Enrichment
- Mix on ice
0.5 uL Short Orange 0.5 uL Short Blue 10 uL DNA/Water 1 uL Klenow 25 uL KAPA Supermix 13 uL Water 50ul total
- Incubate at 30C for 10 min
- Add
0.5ul Orange Primer 0.5ul Blue Primer
- Perform Manufacturer protocol for KAPA
- Cycle the samples in a thermocycler under the following conditions
- 3. Purify the tagged DNA using qiaquick column
- Page Quantification