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Blue:RNA-Seq Experiments:07282014
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==SmartPlus on Mouse Spinal Nuclei (Chun Lab)== Obtained sorted nuclei from Aran: #100 nuclei sorted directly into 2ul clontech dilution buffer (4x) #500 nuclei sorted directly into 2ul clontech dilution buffer (4x) #5000 nuclei sorted into BSA buffer (2x) *stored 100/500 sorts at -80C. Removed 2 of each, vacuum spun down to 1ul for each. *RNA extraction of 5000 using Zymo Micro RNA Kit (including DNAse treatment). Eluted in 6ul and vacuum spun down to 2ul. *Performed SmartPlus protocol as per C1 tube control protocol on 1ul of above samples (only modification is addition of randomer) using 21 cycles for PCR *cDNA Yields: {| {{table}} border | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Sample Concentration (ng/ul)''' |- | NC||0.999 |- | RNA 1||72.1 |- | RNA 2||40.0 |- | 100 nuc 1||7.93 |- | 100 nuc 2||8.75 |- | 500 nuc 1||5.20 |- | 500 nuc 2||6.25 |} * Yields for the RNA 1 and 2 samples are too high (expect more of that seen for the 100-500 nuclei), therefore repeated PCR on RT reaction using 3 less cycles (18 total) {| {{table}} border | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Sample Concentration (ng/ul)''' |- | NC||0.396 |- | RNA 1||11.5 |- | RNA 2||4.34 |} ==NexteraXT== *cDNA diluted to 0.2ng/ul ''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||||||||||12.5ul |- |} ::''Mix and centrifuge'' ::{| {{table}} |- |72C 3 min|||||||||| |- |95C 30s|||||||||| |- |95C 10s -> 55C 30s -> 72C 60s||||||||||12x |- |72C, 5min |- |} *Beads purified (0.8x) twice *Resuspended in 10ul *Ran 1ul on gel: [[File:07282014_PCR1_Gel1.jpg|300px]] {| {{table}} border | align="center" style="background:#f0f0f0;"|'''N7xx''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Barcode''' | align="center" style="background:#f0f0f0;"|'''S5xx''' |- | 1||4960 RNA Extracted (5000 nuc)||CGTGAT||1 |- | 2||5075 RNA Extracted (5000 nuc)||ACATCG||1 |- | 3||4960 - 100 Nuc||CGACTC||1 |- | 4||4960 - 100 Nuc||TGGTCA||1 |- | 5||4960 - 500 Nuc||CACTGT||1 |- | 6||4960 - 500 Nuc||ATTGGC||1 |- | |}
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