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= Normalization of libraries from HL104 run (Library ID: NP-BSPP-Ind1_45-Sep18) to repeat sequencing by GAIIx flowcell = * Continued from the library preparation on 2011/09/18: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-9-18#PCR_Amplification_with_barcoded_primers_.28AmpF6.4Sol_and_AmpR6.3_IndX_No.1-46.29]] * Since the HiSeq at Cancer center (HL104) failed for the second run, and we need to get the data from this set very urgen, Dr. Zhang suggested to do sequencing in our lab by GAIIx. The GAIIx is 1/6 of a Hiseq, so we need to prioritize the libraries to be sequenced and normalized based on the number of reads we got form the first read of HL104. * The prioritized libraries ** Nine libraries from Sergio ** Penn libraries ** Two of HFF libraries ** Three of Hues libraries * Note: Some libraries from Penn have very poor quality and amplification, so I don't have more sample for KEBR028A (B4-plate) and TZHZ018 (F1-plate). For KEBR040 (D4-plate), I have some left, but the total amount will be less than other libraries have been normalized.<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''# of reads''' | align="center" style="background:#f0f0f0;"|'''Indx''' | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''Relative ratio to 4 million reads''' | align="center" style="background:#f0f0f0;"|'''original volume for 60ng''' | align="center" style="background:#f0f0f0;"|'''calculated new volume''' | align="center" style="background:#f0f0f0;"|'''Well ID''' |- | 2,995,193||s_7_1_Indx1||1. H9||A1||0.75||14.35||9.58||A1 |- | 9,894,673||s_7_1_Indx2||**2. H9+Activin 100ng/mL 5days||B1||2.47||50.82||10.27||B1 |- | 7,258,959||s_7_1_Indx3||3. H9+BMP4 50ng/mL 5days||C1||1.81||30.16||8.31||C1 |- | 7,027,403||s_7_1_Indx4||4. HUViPS4F1||D1||1.76||33.61||9.57||D1 |- | 3,708,801||s_7_1_Indx5||5. HUViPS4F1+Activin 100ng/mL 5days||E1||0.93||22.28||12.02||E1 |- | 5,662,790||s_7_1_Indx6||6. HUViPS4F1+BMP4 50ng/mL 5days||F1||1.42||11.63||4.11||F1 |- | 6,962,223||s_7_1_Indx7||7. HUViPS4F3||G1||1.74||32.74||9.40||G1 |- | 6,062,184||s_7_1_Indx8||8. HUViPS4F3+Activin 100ng/mL 5days||H1||1.52||10.85||3.58||H1 |- | 4,602,067||s_7_1_Indx9||9. HUViPS4F3+BMP4 50ng/mL 5days||A3||1.15||20.60||8.95||A3 |- | 3,006,781||s_7_1_Indx32||**2HFF P6||H7||0.75||8.54||5.68||H7 |- | 4,612,655||s_7_1_Indx33||**218 HFF P7||A9||1.15||15.33||6.65||A9 |- | 956,419||s_7_1_Indx34||KEBR028A (B4-plate)||B9||0.24||NA||NA||B9 |- | 2,828,802||s_7_1_Indx36||KEBR040 (D4-plate)||D9||0.71||26.40||18.67||D9 |- | 164,146||s_7_1_Indx37||TZHZ018 (F1-plate)||E9||0.04||NA||NA||E9 |- | 3,210,402||s_7_1_Indx38||TZHZ031A (tube)||F9||0.80||9.45||5.89||F9 |- | 3,996,257||s_7_1_Indx39||TZHZ041A (tube)||G9||1.00||9.44||4.72||G9 |- | 3,205,833||s_7_1_Indx40||TZHZ075 (H01-plate)||H9||0.80||10.59||6.61||H9 |- | 5,605,295||s_7_1_Indx43||**Hues1||C11||1.40||20.71||7.39||C11 |- | 5,028,360||s_7_1_Indx44||**Hues3||D11||1.26||18.50||7.36||D11 |- | 4,495,384||s_7_1_Indx45||**Hues8||E11||1.12||15.01||6.68||E11 |}<br> * Note: I don't have enough Inx2 sample, so I do the PCR to amplify and add to the pool. * Perform PAGE size selection in 5-well 6% TBE gel * Total volume = 145.5ul, add 30ul of 6x loading dye. Loaded 22ul/well (2 gels) [[File:ZhangLab_2 2011-10-17 19hr 18min_PAGE-SS_normBSPPlib.jpg| 500px]]<br> * After gel purification resuspend the libraries in to tal volume 24ul. [[File:ZhangLab_2 2011-10-18 12hr 00min_PQ-BSPP-norm.jpg| 210px]]<br> * 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 6.79 ng/ul or 29.7nM * Concentration was also verified by Qubit ds-DNA HS, conc.: ng/ul or nM * '''Library ID: NP-BSPP_Sep18-norm, HL106 run''' --> PE 110 by GAIIx flowcell * The rest libraries were normalized, pooled and sequenced on 2011/11/16: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-11-15]]
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