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== Capture of differentation samples == ===Capture setup=== {| class="wikitable" cellpadding=1 | align="center" style="background:#f0f0f0;"|'''Tube ID''' | align="center" style="background:#f0f0f0;"|'''Converted sample''' | align="center" style="background:#f0f0f0;"|'''Amount sample (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume sample (uL)''' | align="center" style="background:#f0f0f0;"|'''Volume probes (uL)''' | align="center" style="background:#f0f0f0;"|'''Volume of 10x ampligase buffer''' | align="center" style="background:#f0f0f0;"|'''Volume water (uL)''' | align="center" style="background:#f0f0f0;"|'''Total volume''' | align="center" style="background:#f0f0f0;"|'''Capture sample conc(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Capture probe conc (ng/ul)''' |- | NEG (1)||water||0.0||0.00||4.50||1.00||4.50||10.00||0.00||4.23 |- | N-GM-50 (2)||GM12878||46.5||1.50||4.50||1.50||7.50||15.00||3.10||2.82 |- | N-GM-100 (3)||GM12878||93.0||3.00||9.00||1.50||1.50||15.00||6.20||5.64 |- | N-GM-300 (4)||GM12878||341.0||11.00||15.00||3.00||1.00||30.00||11.37||4.7 |- | MA-16 (5)||H1 p54 Control TeSR||501.6||11.00||15.00||3.00||1.00||30.00||16.72||4.7 |- | MA-17 (6)||H1 p54 Activin-A||469.7||11.00||15.00||3.00||1.00||30.00||15.66||4.7 |- | AP-13 (7)||H1 p47 Control TeSR||314.6||11.00||15.00||3.00||1.00||30.00||10.49||4.7 |- | AP-14 (8)||H1 p47 BMP-4||308.0||11.00||15.00||3.00||1.00||30.00||10.27||4.7 |- | ESC-d0-1 (9)||hESC d0||465.3||11.00||15.00||3.00||1.00||30.00||15.51||4.7 |- | ESC-d0-2 (10)||hESC d0||482.9||11.00||15.00||3.00||1.00||30.00||16.10||4.7 |- | ESC-d0-3 (11)||hESC d0||409.2||11.00||15.00||3.00||1.00||30.00||13.64||4.7 |- | DE-d3-1 (12)||definitive endoderm d3||430.1||11.00||15.00||3.00||1.00||30.00||14.34||4.7 |- | DE-d3-2 (13)||definitive endoderm d3||413.6||11.00||15.00||3.00||1.00||30.00||13.79||4.7 |- | DE-d3-3 (14)||definitive endoderm d3||376.2||11.00||15.00||3.00||1.00||30.00||12.54||4.7 |- | naïve ESC-1 (15)||naïve ESC 1||413.6||11.00||15.00||3.00||1.00||30.00||13.79||4.7 |- | naïve ESC-2 (16)||naïve ESC 2||470.8||11.00||15.00||3.00||1.00||30.00||15.69||4.7 |} ===Low input qPCR, 20 cycles=== * Capture with 50 and 100 ng of template seem to yield the correct band. I might need to amplify for more cycles to increase the band intensity. [[File:HOTSPOTS_lowInput_50,100.bmp | 600px]] [[File:HOTSPOTS_lowInput_50,100_gel.jpg | 300px]] ===Differentiation samples qPCR, 20 cycles === * I performed qPCR for 20 cycles on the samples #4-16, except for #13 and #15 were amplified with 21 cycles. * Each sample was amplified using 28 ul total volume of capture products in 16 v/v%. * Each sample was purified with 1 qiagen column, and eluted with 30 ul water. * Each sample was visualized by gel using 4 ul purified product to quantify prior to pooling Indx29 #13 Indx30 #15 Indx38 #4 Indx39 #5 Indx40 #6 Indx41 #7 Indx42 #8 Indx43 #9 Indx44 #10 Indx45 #11 Indx46 #12 Indx47 #14 Indx48 #16 [[File:HOTSPOTS_differentiation_Song,Sergio.bmp | 600px]] [[File:HOTSPOTS_differentiation_Song,Sergio_gel29-41.jpg | 300px]] [[File:HOTSPOTS_differentiation_Song,Sergio_gel42-48.jpg | 300px]] * Pool #1: 1:1 of each samples Indx29 Indx38 -- DO not pool this library for sequencing because it is positive control, GM12878 sample Indx39 Indx40 Indx41 * Pool #2: 1:1 of each samples Indx42 Indx43 Indx44 Indx45 Indx46 Indx47 Indx48 * Final Pool: 4:7:1 Pool #1 Pool #2 Indx49 (37?) - Repeat of NaiveESC1(capture #15 that failed)
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