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]

File:10.30.12 gel.jpg


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