Noi/NOTES/2014-3-30: Difference between revisions

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>Noi
(Created page with "= DNA methylation assay - MONOD Project, MONOD_V3_12k probe set (BSPP capture) = http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar<...")
 
>Noi
 
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= DNA methylation assay - MONOD Project, MONOD_V3_12k probe set (BSPP capture) =
= DNA methylation assay - MONOD Project, MONOD V3 12k probe set (BSPP capture) =
[[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar]]<br>
[[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar]]<br>
'''Sample list'''<br>
'''Sample list'''<br>
Line 7: Line 7:
: - Blood samples (3 samples)  
: - Blood samples (3 samples)  
* I wanted to compare Hemo KlenTaq and Stoffel Fragment, so I will include one of cancer sample with Stoffel Fragment.
* I wanted to compare Hemo KlenTaq and Stoffel Fragment, so I will include one of cancer sample with Stoffel Fragment.
==== Probe preparation ====
[[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-26]]
==== Sample IDs tracking ====
==== Sample IDs tracking ====
* [http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-2#Sample_IDs_tracking Refer to previous experiment on 2014-03-02]
* [http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-2#Sample_IDs_tracking Refer to previous experiment on 2014-03-02]
* Probe preparation
== Bisulfite conversion ==
== Bisulfite conversion ==
* * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo (lot Mar 2014) and followed the protocol of the kit
* * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo (lot Mar 2014) and followed the protocol of the kit
Line 43: Line 44:
| G10||108.00||5.56||MN_10||14.44||130.00||150.00||G10
| G10||108.00||5.56||MN_10||14.44||130.00||150.00||G10
|-
|-
| G11||82.60||7.26||MN_11||12.74||130.00||150.00||G11
| H11||102.00||5.88||MN_11||14.12||130.00||150.00||G11
|-
|-
| NTC_HemoKlenTaq||0||0||MN_12||20.00||130.00||150.00||NTC
| NTC_HemoKlenTaq||0||0||MN_12||20.00||130.00||150.00||NTC
Line 51: Line 52:
| NTC_Stoffel||0||0||MN_14||20.00||130.00||150.00||NTC
| NTC_Stoffel||0||0||MN_14||20.00||130.00||150.00||NTC
|}
|}
: - Sample G11 was not enough, so I changed to smaple H11.
  '''Program'''  
  '''Program'''  
   
   
Line 56: Line 58:
  2. 54°C for 60 minutes
  2. 54°C for 60 minutes
  3. 4°C hold
  3. 4°C hold
* I elute with Elution buffer 16ul
* I elute with Elution buffer 15ul
* I used 1ul for ssDNA Qubit assay. The volume left after assay ~13ul.
* I used 1ul for ssDNA Qubit assay. The volume left after assay ~13ul.
==== Qubit ss DNA assay ====
==== Qubit ss DNA assay ====
* Dilute 200X (add 1ul of bis-cvt DNA in total 200 assay volume
* Dilute 200X (add 1ul of bis-cvt DNA in total 200 assay volume
{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''uL used'''
| align="center" style="background:#f0f0f0;"|'''Dilution'''
| align="center" style="background:#f0f0f0;"|'''Sample conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Yields in 13ul (ng)'''
| align="center" style="background:#f0f0f0;"|'''% recovery'''
|-
| MN_1||159||ng/mL||1||200||31.90||414.70||69.12
|-
| MN_2||145||ng/mL||1||200||29.10||378.30||63.05
|-
| MN_3||113||ng/mL||1||200||22.50||292.50||48.75
|-
| MN_4||107||ng/mL||1||200||21.40||278.20||46.37
|-
| MN_5||116||ng/mL||1||200||23.10||300.30||50.05
|-
| MN_6||138||ng/mL||1||200||27.60||358.80||59.80
|-
| MN_7||126||ng/mL||1||200||25.10||326.30||54.38
|-
| MN_8||125||ng/mL||1||200||25.00||325.00||54.17
|-
| MN_9||101||ng/mL||1||200||20.20||262.60||43.77
|-
| MN_10||252||ng/mL||1||200||50.50||656.50||109.42*
|-
| MN_11||141||ng/mL||1||200||28.10||365.30||60.88
|-
| MN_13||160||ng/mL||1||200||32.00||416.00||69.33
|}
: - * This sample always has too high recovery rate. This is consistent with previous experiment. I will use double amount for capture since there might be some contaminant causing the background.
== BSPP capture set up ==
== BSPP capture set up ==
=== Probe calculation ===
=== Probe calculation ===
Line 69: Line 106:
| align="center" style="background:#f0f0f0;"|'''Calculation'''
| align="center" style="background:#f0f0f0;"|'''Calculation'''
|-
|-
| Probe size (number of individual probe in oligo mix)||12000||probes||
| Probe size (number of individual probe in oligo mix)||12,000||probes||
|-
|-
| Human bis-cvt gDNA required||200||ng||
| Human bis-cvt gDNA required||250||ng||
|-
|-
| Human gDNA MW||1.822E+12||g/mole||3.0E+09 * 607.4Da/bp
| Human gDNA MW||1.822E+12||g/mole||3.0E+09 * 607.4Da/bp
|-
|-
| Human gDNA( 200ng)||1.098E-19||mole||200E-09g / 1.822E+12g/mole
| Human gDNA( 200ng)||1.372E-19||mole||250E-09g / 1.822E+12g/mole
|-
|-
| Probe required (500:1)||5.488E-17||mole||500 * 1.098E-19mole
| Probe required (500:1)||6.860E-17||mole||500 * 1.098E-19mole
|-
|-
| Probe mix MW (size12,000probes, 110nt)||4.018E+08||g/mole||12,000 probes * (110bp *303.7Da/bp+79Da)
| Probe mix MW (size12,000probes, 110nt)||4.018E+08||g/mole||12,000 probes * (110bp *303.7Da/bp+79Da)
|-
|-
| Mass of probe required||2.205E-08||g||5.488E-17mole * 4.018E+08g/mole
| Mass of probe required||2.757E-08||g||6.860E-17mole * 4.018E+08g/mole
|-
|-
| Mass of probe required||22.05||ng||
| Mass of probe required||27.57||ng||
|-
|-
| Probe conc||||ng/ul||
| Probe conc||12.2||ng/ul||
|-
|-
| Volume of probe||||ul||
| Volume of probe||2.26||ul||
|}
|}
: - I increased bis-DNA to 250ng.
==== Probe & AmpLigase Buffer mix ====
==== Probe & AmpLigase Buffer mix ====
:{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''14.5x'''
|-
| 10X AmpLigase Buffer||2.00||29.00
|-
| MONOD_V3_12k (12.2 ng/ul)||2.26||32.77
|-
| Toal ||4.26||
|}
{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Bis-cvt samples'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Volume for 250ng (ul)'''
| align="center" style="background:#f0f0f0;"|'''Bis-cvt samples'''
| align="center" style="background:#f0f0f0;"|'''H2O (ul)'''
| align="center" style="background:#f0f0f0;"|'''MONOD probe (61.20ng)'''
| align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer'''
| align="center" style="background:#f0f0f0;"|'''Total (ul)'''
| align="center" style="background:#f0f0f0;"|'''Converted DNA amount (ng)'''
|-
| MN_1||31.90||7.84||MN_1||7.90||2.26||2.00||20.00||250.00
|-
| MN_2||29.10||8.59||MN_2||7.15||2.26||2.00||20.00||250.00
|-
| MN_3||22.50||11.11||MN_3||4.63||2.26||2.00||20.00||250.00
|-
| MN_4||21.40||11.68||MN_4||4.06||2.26||2.00||20.00||250.00
|-
| MN_5||23.10||10.82||MN_5||4.92||2.26||2.00||20.00||250.00
|-
| MN_6||27.60||9.06||MN_6||6.68||2.26||2.00||20.00||250.00
|-
| MN_7||25.10||9.96||MN_7||5.78||2.26||2.00||20.00||250.00
|-
| MN_8||25.00||10.00||MN_8||5.74||2.26||2.00||20.00||250.00
|-
| MN_9||20.20||12.38||MN_9||3.36||2.26||2.00||20.00||250.00
|-
| MN_10||50.50||9.90||MN_10||5.84||2.26||2.00||20.00||499.95**
|-
| MN_11||28.10||8.90||MN_11||6.84||2.26||2.00||20.00||250.00
|-
| MN_13||32.00||7.81||MN_13||7.93||2.26||2.00||20.00||250.00
|-
| NTC_HemoKlentaq||0.00||0.00||NTC||15.74||2.26||2.00||20.00||0.00
|-
| NTC_Stoffel||0.00||0.00||NTC_Stoffel||15.74||2.26||2.00||20.00||0.00
|}
'''Program'''
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
==== SLN mix solution (Hemo KlenTaq) ====
:{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Stock conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Final conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Prepare volume (ul) total 125ul'''
|-
| ||||||||||
|-
| Hemo Klentaq||10||U/ul||2||U/ul||25.00
|-
| AmpLigase||5||U/ul||0.5||U/ul||12.50
|-
| dNTP||bgcolor="lawngreen"|10||mM||200||uM||2.50
|-
| 10x AmpLigase Buffer||10||x||1||x||12.50
|-
| H2O||||||||||72.50
|}
: - I prepare extra for MONOD_GP1_V4 capture experiment
==== SLN mix solution (Stoffel Fragment) ====
:{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Stock conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Final conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Prepare volume (ul) total 10ul'''
|-
| ||||||||||
|-
| Stoffel Fragment||10||U/ul||2||U/ul||2.00
|-
| AmpLigase||5||U/ul||0.5||U/ul||1.00
|-
| dNTP||1||mM||200||uM||2.00
|-
| 10x AmpLigase Buffer||10||x||1||x||1.00
|-
| H2O||||||||||4.00
|}
== Amplification ==
: - Add 20% v/v of captured reaction in 100ul PCR (KAPA)
'''Program (Eppendorf Realplex)'''
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
: - Sample MN13 stopped at 20 cycles
: - Sample MN1,3, 6 stopped at 21 cycles
: - Samples MN2,4,5,7-11 stopped at 23 cycles
: - Verify 4ul of PCR product in 6% TBE gel
[[File:ZhangLab_2 2014-04-02 13hr 46min_MONOD_v3capture_gel1.jpg| 400px]] [[File:ZhangLab_2 2014-04-02 13hr 46min_MONOD_v3capture_gel2.jpg|325px]]
: - Purify PCR product with 0.7 volume of AMPure beads
: - Eluted with 60ul H2O
: - Quantify concentration in 6% TBE gel (load 2ul of purified libraries)
[[File:ZhangLab_2 2014-04-02 17hr 21min_MONOD_V3_Seq_PQ_1-8.jpg| 400px]] [[File:ZhangLab_2 2014-04-02 17hr 21min_MONOD_V3_Seq_PQ_9-13.jpg| 250px]]
{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Sample ID'''
| align="center" style="background:#f0f0f0;"|'''Original IDs'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Total amount in 58ul (ng)'''
| align="center" style="background:#f0f0f0;"|'''Volume for  50ng (ul)'''
|-
| MN_1||BXPC3||Ind_1||1.41||81.93||35.39
|-
| MN_2||U87MG||Ind_2||1.12||64.93||44.67
|-
| MN_3||PANC1||Ind_3||1.67||97.11||29.86
|-
| MN_4||BE(2)C||Ind_4||3.13||181.61||15.97
|-
| MN_5||T98G||Ind_5||3.74||216.67||13.38
|-
| MN_6||H9 p43 BMP4||Ind_6||2.13||123.51||23.48
|-
| MN_7||H1 p47 Control||Ind_7||2.96||171.40||16.92
|-
| MN_8||H1 p47 BMP-4||Ind_8||2.86||165.82||17.49
|-
| MN_9||UCLA-B-F10||Ind_9||3.27||189.49||15.30
|-
| MN_10||UCLA-B-G10||Ind_10||2.75||159.39||18.19
|-
| MN_11||UCLA-B-H11||Ind_11||0.91||52.65||55.08
|-
| MN_13||BXPC3_Stoffel||Ind_12||2.68||155.23||18.68
|}
: - Pool equal amount of sequencing libraries (50ng each) and performed PAGE size-selection in 2 of 5-well 6% TBE gels at the band ~375bp
[[File:ZhangLab_2 2014-04-09 11hr 37min_MONOD_V3_SeqLib_PAGESS-1.jpg| 230px]] [[File:ZhangLab_2 2014-04-09 11hr 37min_MONOD_V3_SeqLib_PAGESS-2.jpg| 240px]]
: - Elute with 30ul H2O
: - Verify sequencing libraries in 6% TBE gel by loading 2ul
'''Sequencing ibrary IDs: NP-BSPP-MONOD_V3_Mar30, 2014'''<br>
'''Sequencing run: PE run, 150+7+150 bp'''
: - MiSeq run: 140411_MiSeq (PE 76 + 6 + 76 in stead of PE 150+7+150 bp)

Latest revision as of 00:45, 27 May 2014

DNA methylation assay - MONOD Project, MONOD V3 12k probe set (BSPP capture)[edit]

[Link to calendar]
Sample list

Sample list will be the same as experiment in 2013-03-02
- Cancer cell lines (5 samples)
- Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples)
- Blood samples (3 samples)
  • I wanted to compare Hemo KlenTaq and Stoffel Fragment, so I will include one of cancer sample with Stoffel Fragment.

Probe preparation[edit]

[[1]]

Sample IDs tracking[edit]

Bisulfite conversion[edit]

  • * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo (lot Mar 2014) and followed the protocol of the kit

Bisulfite conversion set up[edit]

Samples Conc. (ng/ul) Volume for 600ng (ul) Go to tube H2O Lightning Conv Reagent Total (ul) Samples
CANC_1 47.70 12.58 MN_1 7.42 130.00 150.00 CANC_1
CANC_2 45.40 13.22 MN_2 6.78 130.00 150.00 CANC_2
CANC_3 41.40 14.49 MN_3 5.51 130.00 150.00 CANC_3
CANC_4 47.10 12.74 MN_4 7.26 130.00 150.00 CANC_4
CANC_5 68.20 8.80 MN_5 11.20 130.00 150.00 CANC_5
SR-3 62.20 9.65 MN_6 10.35 130.00 150.00 SR-3
MA-16 85.00 7.06 MN_7 12.94 130.00 150.00 MA-16
MA-17 49.40 12.15 MN_8 7.85 130.00 150.00 MA-17
F10 106.00 5.66 MN_9 14.34 130.00 150.00 F10
G10 108.00 5.56 MN_10 14.44 130.00 150.00 G10
H11 102.00 5.88 MN_11 14.12 130.00 150.00 G11
NTC_HemoKlenTaq 0 0 MN_12 20.00 130.00 150.00 NTC
CANC_1 47.70 12.58 MN_13 7.42 130.00 150.00 CANC_1
NTC_Stoffel 0 0 MN_14 20.00 130.00 150.00 NTC
- Sample G11 was not enough, so I changed to smaple H11.
Program 

1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
  • I elute with Elution buffer 15ul
  • I used 1ul for ssDNA Qubit assay. The volume left after assay ~13ul.

Qubit ss DNA assay[edit]

  • Dilute 200X (add 1ul of bis-cvt DNA in total 200 assay volume
Sample Conc. in the Qubit Unit uL used Dilution Sample conc. (ng/ul) Yields in 13ul (ng) % recovery
MN_1 159 ng/mL 1 200 31.90 414.70 69.12
MN_2 145 ng/mL 1 200 29.10 378.30 63.05
MN_3 113 ng/mL 1 200 22.50 292.50 48.75
MN_4 107 ng/mL 1 200 21.40 278.20 46.37
MN_5 116 ng/mL 1 200 23.10 300.30 50.05
MN_6 138 ng/mL 1 200 27.60 358.80 59.80
MN_7 126 ng/mL 1 200 25.10 326.30 54.38
MN_8 125 ng/mL 1 200 25.00 325.00 54.17
MN_9 101 ng/mL 1 200 20.20 262.60 43.77
MN_10 252 ng/mL 1 200 50.50 656.50 109.42*
MN_11 141 ng/mL 1 200 28.10 365.30 60.88
MN_13 160 ng/mL 1 200 32.00 416.00 69.33
- * This sample always has too high recovery rate. This is consistent with previous experiment. I will use double amount for capture since there might be some contaminant causing the background.

BSPP capture set up[edit]

Probe calculation[edit]

MONOD_V3_12k, size 12,000

Probe:taeget 500:1 Unit Calculation
Probe size (number of individual probe in oligo mix) 12,000 probes
Human bis-cvt gDNA required 250 ng
Human gDNA MW 1.822E+12 g/mole 3.0E+09 * 607.4Da/bp
Human gDNA( 200ng) 1.372E-19 mole 250E-09g / 1.822E+12g/mole
Probe required (500:1) 6.860E-17 mole 500 * 1.098E-19mole
Probe mix MW (size12,000probes, 110nt) 4.018E+08 g/mole 12,000 probes * (110bp *303.7Da/bp+79Da)
Mass of probe required 2.757E-08 g 6.860E-17mole * 4.018E+08g/mole
Mass of probe required 27.57 ng
Probe conc 12.2 ng/ul
Volume of probe 2.26 ul
- I increased bis-DNA to 250ng.

Probe & AmpLigase Buffer mix[edit]

Components Volume (ul) 14.5x
10X AmpLigase Buffer 2.00 29.00
MONOD_V3_12k (12.2 ng/ul) 2.26 32.77
Toal 4.26
Bis-cvt samples Conc. (ng/ul) Volume for 250ng (ul) Bis-cvt samples H2O (ul) MONOD probe (61.20ng) 10X AmpLigase Buffer Total (ul) Converted DNA amount (ng)
MN_1 31.90 7.84 MN_1 7.90 2.26 2.00 20.00 250.00
MN_2 29.10 8.59 MN_2 7.15 2.26 2.00 20.00 250.00
MN_3 22.50 11.11 MN_3 4.63 2.26 2.00 20.00 250.00
MN_4 21.40 11.68 MN_4 4.06 2.26 2.00 20.00 250.00
MN_5 23.10 10.82 MN_5 4.92 2.26 2.00 20.00 250.00
MN_6 27.60 9.06 MN_6 6.68 2.26 2.00 20.00 250.00
MN_7 25.10 9.96 MN_7 5.78 2.26 2.00 20.00 250.00
MN_8 25.00 10.00 MN_8 5.74 2.26 2.00 20.00 250.00
MN_9 20.20 12.38 MN_9 3.36 2.26 2.00 20.00 250.00
MN_10 50.50 9.90 MN_10 5.84 2.26 2.00 20.00 499.95**
MN_11 28.10 8.90 MN_11 6.84 2.26 2.00 20.00 250.00
MN_13 32.00 7.81 MN_13 7.93 2.26 2.00 20.00 250.00
NTC_HemoKlentaq 0.00 0.00 NTC 15.74 2.26 2.00 20.00 0.00
NTC_Stoffel 0.00 0.00 NTC_Stoffel 15.74 2.26 2.00 20.00 0.00
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix solution (Hemo KlenTaq)[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul) total 125ul
Hemo Klentaq 10 U/ul 2 U/ul 25.00
AmpLigase 5 U/ul 0.5 U/ul 12.50
dNTP 10 mM 200 uM 2.50
10x AmpLigase Buffer 10 x 1 x 12.50
H2O 72.50
- I prepare extra for MONOD_GP1_V4 capture experiment

SLN mix solution (Stoffel Fragment)[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul) total 10ul
Stoffel Fragment 10 U/ul 2 U/ul 2.00
AmpLigase 5 U/ul 0.5 U/ul 1.00
dNTP 1 mM 200 uM 2.00
10x AmpLigase Buffer 10 x 1 x 1.00
H2O 4.00

Amplification[edit]

- Add 20% v/v of captured reaction in 100ul PCR (KAPA)
Program (Eppendorf Realplex)
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- Sample MN13 stopped at 20 cycles
- Sample MN1,3, 6 stopped at 21 cycles
- Samples MN2,4,5,7-11 stopped at 23 cycles
- Verify 4ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-04-02 13hr 46min MONOD v3capture gel1.jpg File:ZhangLab 2 2014-04-02 13hr 46min MONOD v3capture gel2.jpg
- Purify PCR product with 0.7 volume of AMPure beads
- Eluted with 60ul H2O
- Quantify concentration in 6% TBE gel (load 2ul of purified libraries)
File:ZhangLab 2 2014-04-02 17hr 21min MONOD V3 Seq PQ 1-8.jpg File:ZhangLab 2 2014-04-02 17hr 21min MONOD V3 Seq PQ 9-13.jpg
Sample ID Original IDs Index Conc. (ng/ul) Total amount in 58ul (ng) Volume for 50ng (ul)
MN_1 BXPC3 Ind_1 1.41 81.93 35.39
MN_2 U87MG Ind_2 1.12 64.93 44.67
MN_3 PANC1 Ind_3 1.67 97.11 29.86
MN_4 BE(2)C Ind_4 3.13 181.61 15.97
MN_5 T98G Ind_5 3.74 216.67 13.38
MN_6 H9 p43 BMP4 Ind_6 2.13 123.51 23.48
MN_7 H1 p47 Control Ind_7 2.96 171.40 16.92
MN_8 H1 p47 BMP-4 Ind_8 2.86 165.82 17.49
MN_9 UCLA-B-F10 Ind_9 3.27 189.49 15.30
MN_10 UCLA-B-G10 Ind_10 2.75 159.39 18.19
MN_11 UCLA-B-H11 Ind_11 0.91 52.65 55.08
MN_13 BXPC3_Stoffel Ind_12 2.68 155.23 18.68
- Pool equal amount of sequencing libraries (50ng each) and performed PAGE size-selection in 2 of 5-well 6% TBE gels at the band ~375bp
File:ZhangLab 2 2014-04-09 11hr 37min MONOD V3 SeqLib PAGESS-1.jpg File:ZhangLab 2 2014-04-09 11hr 37min MONOD V3 SeqLib PAGESS-2.jpg
- Elute with 30ul H2O
- Verify sequencing libraries in 6% TBE gel by loading 2ul

Sequencing ibrary IDs: NP-BSPP-MONOD_V3_Mar30, 2014
Sequencing run: PE run, 150+7+150 bp

- MiSeq run: 140411_MiSeq (PE 76 + 6 + 76 in stead of PE 150+7+150 bp)