Rui:LabNotes/SingleCell/2012-10-30
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2nd half[edit]
- Continue with 10.29.12 notes [1]
- split in 1ul (~10ng 1/10 of the 100ng RNA input) and the rest (~4ul, close to 50ng used in 10.29.12)
- test if the PCR cycle and yield is proportional with the cDNA input - good dsDNA amplification
- estimate the Ct, saturation cycles/final yields based on RNA input
- with the ~4ul cDNA, amplify to saturation and reach 500ng total
QPCR curve[edit]
- Stop the reaction when it reaches saturation / Flur. above 0.7
- 2,5,9,11: stop at 16 ct
- others: stop at 18 ct
- 4,7: stop at 20 ct
4ul_16ct File:10.30.12 16ct.jpg 4ul_18ct File:10.30.12 18ct.jpg
1ul_20ct File:10.30.12 20ct.jpg
Gel[edit]
ChrX capture[edit]
- pre-capture preparation and protocol can be found in Alice's notes: [Link]
Post capture amplification[edit]
- Bead purification (1.2X) with 1.5ml tubes (9 samples), elute in 40ul
- PCR with SyF and SyR in 100ul
DNA 20ul SYBR green 0.4ul 2X Phushion MM 50ul SyF (100uM) 1ul SyR (100uM) 1ul Fill up with water to 100ul
98C, 30sec 98C, 10sec 60C, 30sec 72C, 30sec repeat 11 cycles 72C, 3min
- Beads purification (0.8X), elute with 12ul, 1ul for gel
Final check[edit]
File:11.6.12 KE totoRNA QPCR.jpgFile:11.6.12 KE totoRNA TBE.jpg
Sequencing primer design[edit]
- Sequencing library
AATGATACGGCGACCACCGAGATCT_ACACTCTTTCCCTACACGACG_TT_N10_BC8_rGrGrG======|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||======VNT30_TCTAGCCTTCTC_GCCAAGTCGTCCTTACGGCTC_Indx_TAGAGCATACGGCAGAAGACGAAC
- original illumina seq. primers
Read1: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT Read2:
- New designed Read1 primer for totoRNAseq libraries
' | Sequence | length | IDT, Tm | Kun, Tm |
Seq. Read 1_base | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGTT | 48 | 69 | 77.85 |
test1 | CGACCACCGAGATCTACACTCTTTCCCTACACGACGTT | 38 | 66 | 74 |
test2 | CCACCGAGATCTACACTCTTTCCCTACACGACGTT | 35 | 65 | 72.82 |
totoRNA_Seq. Read 1 | CACCGAGATCTACACTCTTTCCCTACACGACGTT | 34 | 64 | 71.72 |