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==PBMC C1 Experiment== Extracted RNA from bulk samples using Zymo microRNA Kit and eluted in 6ul. Determined [RNA] using Qubit: #CD4+ 26.1 ng/uL #CD19+ 6.27 ng/uL #PBMC 95.8 ng/uL Diluted to 1ng/ul and used 1ng as tube control for C1 run 20140702 (followed standard Smart+randomer C1 tube control protocol with 3 fewer cycles in last stage of PCR) Qubit quantification of PCR product: #CD4+ 6.56ng/ul - dilute 1:22 for 0.3ng/ul #CD19+ 7.60ng/ul - dilute 1:25 for 0.3ng/ul #PBMC 5.02ng/ul - dilute 1:17 for 0.3ng/ul #Neg Cont 1.64ng/ul ==NexteraXT== *Single nuclei cDNA (generated on C1 by Rui 05242014) transferred to new 96-well plate using EpiMotion *cDNA for single nuclei was used undiluted for NexteraXT: ''Nextera Tagmentation'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagment DNA Buffer||||||||||2.5ul |- |Amplification Tagment Mix||||||||||1.25ul |- |Diluted Sample||||||||||1.25ul |- |Total||||||||||5ul |- |} ::*Vortex and spin ::*Incubate 55C 10 min ::*Hold 10C ::*Once sample reaches 10C add 1.25ul of NT buffer in block ::*Vortex and spin ''Nextera Amplification'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagmented Sample||||||||||6.25ul |- |Nextera PCR Mix||||||||||3.75ul |- |S501 Primer||||||||||1.25ul |- |N7id(1-24) Primer||||||||||1.25ul |- |Total||||||||||10ul |- |} ::''Mix and centrifuge'' ::{| {{table}} |- |72C 3 min|||||||||| |- |95C 30s|||||||||| |- |95C 10s -> 55C 30s -> 72C 60s||||||||||12x |- |72C, 5min |- |} *Combined samples 1-20 (group 1 with N7 ids 1-20) *Combined samples 21-40 (group 2 with N7 ids 1-20) *Combined tube controls *Beads purified (0.8x) twice *Resuspended Group 1 and Group 2 in 50ul; resuspended tube controls in 10ul *Ran 1ul on gel: [[File:07022014_PCR1_Gel1.jpg]] ==Library Summary== {| {{table}} border | align="center" style="background:#f0f0f0;"|'''Group''' | align="center" style="background:#f0f0f0;"|'''Sample Name''' | align="center" style="background:#f0f0f0;"|'''Nextera_Ind''' |- | 1||1_PBMC_C03||1 |- | 1||1_PBMC_C09||2 |- | 1||1_PBMC_C15||3 |- | 1||1_PBMC_C25||4 |- | 1||1_PBMC_C43||5 |- | 1||1_PBMC_C14||6 |- | 1||1_PBMC_C20||7 |- | 1||1_PBMC_C26||8 |- | 1||1_PBMC_C01||9 |- | 1||1_PBMC_C39||10 |- | 1||1_PBMC_C61||11 |- | 1||1_PBMC_C87||12 |- | 1||1_PBMC_C56||13 |- | 1||1_PBMC_C68||14 |- | 1||1_PBMC_C74||15 |- | 1||1_PBMC_C80||16 |- | 1||1_PBMC_C92||17 |- | 1||1_PBMC_C51||18 |- | 1||1_PBMC_C57||19 |- | 1||1_PBMC_C63||20 |- | 2||1_PBMC_C69||1 |- | 2||1_PBMC_C06||2 |- | 2||1_PBMC_C18||3 |- | 2||1_PBMC_C24||4 |- | 2||1_PBMC_C28||5 |- | 2||1_PBMC_C34||6 |- | 2||1_PBMC_C46||7 |- | 2||1_PBMC_C17||8 |- | 2||1_PBMC_C35|||9 |- | 2||1_PBMC_C41||10 |- | 2||1_PBMC_C04||11 |- | 2||1_PBMC_C48||12 |- | 2||1_PBMC_C64||13 |- | 2||1_PBMC_C78||14 |- | 2||1_PBMC_C90||15 |- | 2||1_PBMC_C53||16 |- | 2||1_PBMC_C65||17 |- | 2||1_PBMC_C54||18 |- | 2||1_PBMC_C88||19 |- | 2||1_PBMC_C94||20 |- | TubeCont||CD4+||21 |- | TubeCont||CD19+||22 |- | TubeCont||PBMC||23 |- |}
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