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== Low input (1ng) library preparation using Kapa kit (Continued) == * The main point of doing this experiment is to try preparing sequencing library of Jeff's samples using ligation-based method. However, it didn't work well, and I couldn't get it work for the positive control of 1ng DNA input. There could be the limitation of DNA input and the adapter to template ratio. I would do experiment by varying amount of DNA input of 1, 2, 5 and 10ng, and also included 2 of Jeff' samples. * I prepared DNA for shearing using NA12878 samples * Confirm concentration by dsDNA Qubit Quantification (100ng/ul). By using Nanodrop the concentration is 219ng/ul * Dilute DNA to 1ng/ul (2ul of 100ng/ul in total volume 200ul with H2O) * Dilute to different concentration and aliquot 50ul into microtube {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''1ng''' | align="center" style="background:#f0f0f0;"|'''2ng''' | align="center" style="background:#f0f0f0;"|'''5ng''' | align="center" style="background:#f0f0f0;"|'''10ng''' |- | DNA 1ng/ul||2.20||4.40||11.00||22.00 |- | H2O||107.80||105.60||99.00||88.00 |- | Total amount in 50ul (ng)||1.00||2.00||5.00||10.00 |} * Sheared DNA into 400bp fragments * Performed library prep using the same procedure as I did on April 12, 2013. Use amount of adapter calculated for 10ng DNA template (20:1) for all samples. This amount of adapters gave different ratio of adapter: template for differen input -> 200:1 for 1ng, 100:1 for 2ng, 40:1 for 5ng and 20:1 for 10ng. * '''Note:''' I normally used 20:1 for ligation with minimum DNA input 100ng * Samples: NTC, 1ng, 2ng, 5ng, 10ng, Jeff #4 and Jeff #5 * After ligation, eluted with 20ul H2O and use all for qPCR. {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''8.5 rxn mix''' |- | adapter ligated DNA||20.00||0.00 |- | PCR_F(10uM)||1.00||8.50 |- | PCR_R.N2IndX(10uM)||1.00||0.00 |- | H2O||28.00||238.00 |- | 2X KAPA SYBR MM||50.00||425.00 |- | Total volume||100.00||850.00 |} * Aliquot 79ul, add 1ul PCR_R.N2.Ind, 20ul template '''Program''' 98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x20, 72C - 2min, hold at 15C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | 1ng||PCR_R.N2Indx1 |- | 2ng||PCR_R.N2Indx2 |- | 5ng||PCR_R.N2Indx3 |- | 10ng||PCR_R.N2Indx4 |- | Jeff#4||PCR_R.N2Indx5 |- | Jeff#5||PCR_R.N2Indx6 |- | NTC2||PCR_R.N2Indx7 |- | NTC1||PCR_R.N2Indx8 |} [[File:20130416_qPCR_LowInput_varyinput.png| 550px]] * Note: The qPCR curve of 2ng and 5ng were pretty much the same, but the signal of 2ng came from the adapter as the intensity of ~150bp band was stronger than the 5ng reaction. * PAGE analysis to verify the PCR products by loading 5ul of PCR product without bead purification '''Loaded 5ul of PCR products''' [[File:ZhangLab_2 2013-04-16 19hr 25min_5ul.jpg| 350px]] '''Loaded 15ul of PCR products''' [[File:ZhangLab_2 2013-04-16 20hr 17min_15ul.jpg| 380px]] '''Note:''' NTC1 = NTC without adapters (I only added H2O in qPCR, NTC2 = NTC with adapters (from the beginning steps) * From the image,I saw amplification in 5ng and 10ng, I barely saw the signal in 2ng. I loaded more of product (15ul), but the signal in 2ng was still very faint. There is no amplification in Jeff' sample and 1ng control. It clearly showed that the amount of DNA input as low as 1ng is the case that the library prep dind't work in my hands.This may be the reason that I could not prepare library using Jeff' samples which have very low amount of DNA input. * Need suggestion and discusion from Dr. Zhang and Jeff based on these results. === 2013_04_18 === * Dr. Zhang suggested Jeff to try CGI protocol by using dUTP, and suggested me to checked with Rui for the concentration of adapter she used for her mouse PGC RNAseq experiment. * Note from Rui: email on April 18, 2013 ** http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/SingleCellExpr/2011-7-5#PCR_products ** http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-11#End repair ** http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-11#Pre-run ** http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-12#Prepare_RNAseq_library_for_sequencing ** Summary of adapter she used (note: her fragment size ~200bp, mine is 400bp) *** For template <10ng: added 0.08ul of 30uM adapters --> final conc. in 50ul = 0.048uM. This means that if I used 20uM adapters, I should add (50ul*0.048uM)/50ul = 0.12ul *** For template 10ng - 50ng: added 0.4ul of 30uM adapters --> final conc. in 50ul = 0.24uM (5X more than template <10ng) ** Perform qPCR in 50ul reaction, and added 1ul of 10uM each primer. * I have some discussions with Jeff, and he has shared this paper for CGI protocol as Dr. Zhang suggested [http://www.nature.com/nature/journal/v487/n7406/full/nature11236.html]. * I am planning to repeat experiment using low DNA input (1ng, 2ng and 5ng input) and adapter concentration folowing Rui's experiment. I still have sheared gDNA of NA12878 (may need to re-quantify with Qubit assay again to confirm conc.) * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-24
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