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===Results=== *FACS results, ~10-15% of events were nuclei. was probably dirty from mixing with mouse ES cells which were plated on gelatin and had to be brought up with accutase [[File:Batch Analysis 2015 12 16.tiff|600px]] *plate layout for i5/i7 barcodes {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''N701''' | align="center" style="background:#f0f0f0;"|'''N702''' | align="center" style="background:#f0f0f0;"|'''N703''' | align="center" style="background:#f0f0f0;"|'''N704''' | align="center" style="background:#f0f0f0;"|'''N705''' | align="center" style="background:#f0f0f0;"|'''N706''' | align="center" style="background:#f0f0f0;"|'''N707''' | align="center" style="background:#f0f0f0;"|'''N710''' | align="center" style="background:#f0f0f0;"|'''N711''' | align="center" style="background:#f0f0f0;"|'''N712''' | align="center" style="background:#f0f0f0;"|'''N714''' | align="center" style="background:#f0f0f0;"|'''N715''' |- | ||||1||2||3||4||5||6||7||8||9||10||11||12 |- | scT7_S502||A||A1, scT7_S502, N701||A2, scT7_S502, N702||A3, scT7_S502, N703||A4, scT7_S502, N704||A5, scT7_S502, N705||A6, scT7_S502, N706||A7, scT7_S502, N707||A8, scT7_S502, N710||A9, scT7_S502, N711||A10, scT7_S502, N712||A11, scT7_S502, N714||A12, scT7_S502, N715 |- | scT7_S503||B||B1, scT7_S503, N701||B2, scT7_S503, N702||B3, scT7_S503, N703||B4, scT7_S503, N704||B5, scT7_S503, N705||B6, scT7_S503, N706||B7, scT7_S503, N707||B8, scT7_S503, N710||B9, scT7_S503, N711||B10, scT7_S503, N712||B11, scT7_S503, N714||B12, scT7_S503, N715 |- | scT7_S505||C||C1, scT7_S505, N701||C2, scT7_S505, N702||C3, scT7_S505, N703||C4, scT7_S505, N704||C5, scT7_S505, N705||C6, scT7_S505, N706||C7, scT7_S505, N707||C8, scT7_S505, N710||C9, scT7_S505, N711||C10, scT7_S505, N712||C11, scT7_S505, N714||C12, scT7_S505, N715 |- | scT7_S506||D||D1, scT7_S506, N701||D2, scT7_S506, N702||D3, scT7_S506, N703||D4, scT7_S506, N704||D5, scT7_S506, N705||D6, scT7_S506, N706||D7, scT7_S506, N707||D8, scT7_S506, N710||D9, scT7_S506, N711||D10, scT7_S506, N712||D11, scT7_S506, N714||D12, scT7_S506, N715 |- | scT7_S507||E||E1, scT7_S507, N701||E2, scT7_S507, N702||E3, scT7_S507, N703||E4, scT7_S507, N704||E5, scT7_S507, N705||E6, scT7_S507, N706||E7, scT7_S507, N707||E8, scT7_S507, N710||E9, scT7_S507, N711||E10, scT7_S507, N712||E11, scT7_S507, N714||E12, scT7_S507, N715 |- | scT7_S508||F||F1, scT7_S508, N701||F2, scT7_S508, N702||F3, scT7_S508, N703||F4, scT7_S508, N704||F5, scT7_S508, N705||F6, scT7_S508, N706||F7, scT7_S508, N707||F8, scT7_S508, N710||F9, scT7_S508, N711||F10, scT7_S508, N712||F11, scT7_S508, N714||F12, scT7_S508, N715 |- | scT7_S510||G||G1, scT7_S510, N701||G2, scT7_S510, N702||G3, scT7_S510, N703||G4, scT7_S510, N704||G5, scT7_S510, N705||G6, scT7_S510, N706||G7, scT7_S510, N707||G8, scT7_S510, N710||G9, scT7_S510, N711||G10, scT7_S510, N712||G11, scT7_S510, N714||G12, scT7_S510, N715 |- | scT7_S511||H||H1, scT7_S511, N701||H2, scT7_S511, N702||H3, scT7_S511, N703||H4, scT7_S511, N704||H5, scT7_S511, N705||H6, scT7_S511, N706||H7, scT7_S511, N707||H8, scT7_S511, N710||H9, scT7_S511, N711||H10, scT7_S511, N712||H11, scT7_S511, N714||H12, scT7_S511, N715 |- | |} *TBU after IVT, single cell combinatorial indexing *A12 - D12. 25 human GM12878 / mouse F123 cells, 10 uL (whole reaction for gel) *E12. 500 human GM12878 / mouse F123 cells, 1 uL for gel *G12-H12. NTC, FACS sheath buffer, whole reaction for gel {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''total''' |- | ||4532.763797||5|| |- | ||2132.932458||2.5|| |- | A12. 25 cells||783.5846339||1.094330566||1.094330566 |- | B12. 25 cells||1151.700704||1.477811755||1.477811755 |- | C12. 25 cells||1807.649147||2.161139404||2.161139404 |- | D12. 25 cells||1616.98589||1.962517887||1.962517887 |- | E12. 500 cells||4174.430366||4.626709776||46.26709776 |- | F12 500 cells||3242.147971||3.65551403||36.5551403 |- | G12. NTC||390.1517508||0.684475843||0.684475843 |- | H12. NTC||44.51531534||0.324412586||0.324412586 |- | |} *gel *whole reaction for NTC is 10 uL [[File:ZhangLab 2 2015-12-17 19hr 23min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-17 19hr 25min-labeled.jpg|600px]] '''Test on six 25 cell samples and controls''' *worked well so will process the rest of the samples *PCR curves [[File:2015-12-18 scTHS-seq RNA processing test libraries.bmp|600px]] *gel quant {| {{table}} | align="center" style="background:#f0f0f0;"|'''12/23/2015''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||||2499.615864||5|||||||| |- | ||||1487.083729||2.5||for 50 ng||*2|||| |- | 25 cells||A11||6607.170395||15.14178807||3.30211992||6.604239841|||| |- | 25 cells||B11||11910.86456||28.2369136||1.77073177||3.54146354|||| |- | 25 cells||C11||9360.151844||21.93905739||2.279040485||4.558080971|||| |- | 25 cells||D11||24062.18975||58.23923342||0.858527784||1.717055568|||| |- | 25 cells||E11||19480.27317||46.92621822||1.06550244||2.131004879|||| |- | 25 cells||F11||4519.732545||9.987784116||5.006115413||10.01223083|||| |- | 500 cells||E12||29587.9675||71.88269609||0.695577694||1.391155389|||| |- | 500 cells||F12||33092.05225||80.53448259||0.620852067||1.241704135||sum||35.19693515 |- | NTC||G11||348.1816111||-0.312014744||1||2||dye||7.039387029 |- | NTC||H11||119.1824496||-0.877426827||1||2|||| |- | ||||||||#DIV/0!|||||| |- | ||||||||#DIV/0!|||||| |- | |} [[File:ZhangLab 2 2015-12-21 11hr 29min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-21 11hr 30min-invert-labeled.jpg|600px]] '''Processing of the rest of the samples, RNA processing and PCR barcoding''' *the qPCR machine gave a "voltage error" after 8 cycles, so restarted the machine and ran another 7 cycles. the libraries look a little over amplified, and look like the 500 cell samples when run for 15 cycles, when they only needed ~9 cycles. However should be fine, will run mi-seq and test. *PCR curves [[File:2015-12-22 scTHS-seq RNA processing libraries rest1.bmp|600px]] [[File:2015-12-22 scTHS-seq RNA processing libraries rest2.bmp|600px]] *gel quant {| {{table}} | align="center" style="background:#f0f0f0;"|'''12/23/2016''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||intensity||ng/ul|||||||||| |- | ||4935.006301||5|||||||||| |- | ||1749.18236||2.5||size select amt||||dye||total|| |- | A1||5682.233182||5.586368625||2.685107448||||22.4||135.2313129|| |- | B1||3769.715259||4.085565411||3.671462451||||||27.04626257|| |- | C1||2195.742785||2.850427734||5.262368108|||||||| |- | D1||4118.312926||4.359119187||3.44106214|||||||| |- | E1||2199.033371||2.853009943||5.257605231|||||||| |- | A3||7215.256742||6.789372611||2.209335215|||||||| |- | B3||10046.63837||9.011232391||1.664589187|||||||| |- | C3||10596.09843||9.442408178||1.588577799|||||||| |- | D3||8162.056185||7.532351084||1.991410097|||||||| |- | E3||8246.830999||7.598876114||1.973976122|||||||| |- | F3||6387.908015||6.140130262||2.442944915|||||||| |- | A2||5409.844933||5.372618388||2.791934755|||||||| |- | B2||9281.993181||8.411195158||1.78333753|||||||| |- | C2||13239.02703||11.5163839||1.302492183|||||||| |- | D2||9393.517292||8.498711067||1.764973522|||||||| |- | E2||9944.62984||8.931183606||1.679508637|||||||| |- | A4||17050.01723||14.50696833||1.033985851|||||||| |- | B4||10925.13647||9.700612994||1.546294034|||||||| |- | C4||6187.382425||5.982772547||2.507198775|||||||| |- | D4||4867.636245||4.947132941||3.03205921|||||||| |- | E4||8330.122738||7.664237337||1.957141897|||||||| |- | F4||11681.23458||10.29394311||1.457167564|||||||| |- | A5||10801.60806||9.603677001||1.561901759|||||||| |- | B5||11984.79584||10.53215563||1.42420987|||||||| |- | C5||8062.811208||7.454470935||2.012215237|||||||| |- | D5||15156.9111||13.02139806||1.151950039|||||||| |- | E5||6747.921491||6.422642324||2.335487365|||||||| |- | F5||22632.00551||18.88730164||0.794184383|||||||| |- | A6||10050.21524||9.014039248||1.664070855|||||||| |- | B6||8686.149373||7.943620819||1.888307655|||||||| |- | C6||9057.964953||8.235394304||1.821406413|||||||| |- | D6||13225.85546||11.50604782||1.303662234|||||||| |- | E6||14710.69322||12.67123914||1.183783199|||||||| |- | F6||12971.15925||11.30618099||1.326707932|||||||| |- | A7||7431.701526||6.959222536||2.155413183|||||||| |- | B7||7796.667885||7.245621256||2.070215855|||||||| |- | C7||9782.658031||8.804080059||1.70375552|||||||| |- | D7||9693.692898||8.734266774||1.717373695|||||||| |- | E7||9005.73766||8.19441017||1.830516131|||||||| |- | F7||3500.129061||3.874014018||3.871952949|||||||| |- | A8||8856.770391||8.077511628||1.85700754|||||||| |- | B8||14507.68891||12.51193631||1.198855208|||||||| |- | C8||14470.49739||12.48275114||1.201658179|||||||| |- | D8||10944.94159||9.716154598||1.543820639|||||||| |- | E8||10033.13916||9.000639201||1.666548304|||||||| |- | F8||7992.216314||7.399073262||2.027280913|||||||| |- | A9||13946.39042||12.07147058||1.242599226|||||||| |- | B9||8733.84401||7.981048058||1.879452409|||||||| |- | C9||7726.95023||7.190911975||2.085966294|||||||| |- | D9||10083.42655||9.040101042||1.659273489|||||||| |- | E9||12962.49664||11.29938321||1.327506088|||||||| |- | F9||10232.93155||9.157421559||1.638015669|||||||| |- | A10||20984.12112||17.59416333||0.852555459|||||||| |- | B10||17957.82029||15.21934531||0.98558773|||||||| |- | C10||12757.44145||11.138471||1.346683939|||||||| |- | D10||4397.735007||4.578389057||3.276261543|||||||| |- | E10||17135.02547||14.57367654||1.02925298|||||||| |- | F10||15165.6988||13.028294||1.151340306|||||||| |- | |} *gel images [[File:ZhangLab 2 2015-12-23 11hr 56min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 11hr 59min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 12hr 01min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 12hr 02min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 13hr 05min-lableled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 13hr 06min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 13hr 07min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 13hr 08min-labeled.jpg|600px]] *gel size selection [[File:ZhangLab 2 2015-12-23 16hr 16min-labeled.jpg|600px]] [[File:ZhangLab 2 2015-12-23 16hr 19min-labeled.jpg|600px]] *get PCR curve images *after gel size selection of libraries [[File:ZhangLab 2 2015-12-23 19hr 38min-labeled.jpg|600px]]
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