Rui:LabNotes/SingleCell/2012-12-17

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Hs Ref RNA with totoRNAseq ranging from 1ng to 10pg[edit]

  • Repeat procedure used on 11.8.12/11.10.12
  • set 2 condition prior to RT: 72C 3min vs 10C 10min
  • set 2 condition for TSO: TSO.r02 vs TSO.idXX

Samples and procedure[edit]

  • PNK: 1:2d in water
  • PAP:1:4d in water
  • RNaseIn: 1:2d in water
  • MMLV:1:2d in water
' RNA Frag. T20.id ' Anneal after RT TSO N2.id
X2 1ng 1min 1uM id01-02 72C, 3min 1uM
X2 NTC 1min 1uM id07-08 72C, 3min 3.2ng TSO.r02 N2.id01
X12 100pg 1min 1uM others to 16 72C, 3min 1.6ng X 2 TSO.id02 N2.id02
X2 1ng 1min 1uM id01-02 10C,15min 1uM
X2 NTC 1min 1uM id07-08 10C,15min 3.2ng TSO.r02 N2.id03
X12 100pg 1min 1uM others to 16 10C,15min 1.6ng X 2 TSO.id04 N2.id04
X2 100pg 1min 0.1uM id01-02 72C, 3min 1uM
X2 NTC 1min 0.1uM id07-08 72C, 3min 320pg TSO.r02 N2.id05
X12 10pg 1min 0.1uM others to 16 72C, 3min 0.16ng X 2 TSO.id06 N2.id06
X2 100pg 1min 0.1uM id01-02 10C,15min 1uM
X2 NTC 1min 0.1uM id07-08 10C,15min 320pg TSO.r02 N2.id07
X12 10pg 1min 0.1uM others to 16 10C,15min 0.16ng X 2 TSO.id08 N2.id08

Results[edit]

File:12.17.12 1-4.jpgFile:12.17.12 5-8.jpg

File:12.17.12 gel.jpg

Size selection and re-amplification[edit]

  • Directly load combine 1-2, 3-4, 5-6 and 7-8 for size selection
  • Gel was pooled into 4 tubes:
100pg: >300, <300
10pg: >300, <300
  • EtOH precipitation, resuspend in 11ul, 1ul for Qubit
#1.10pg,<300bp	        2.60ng/ul     26ng
#2.10pg,>300bp	        1.45ng/ul     14.5ng	
#3.100pg,<300bp	10.7ng/ul     107ng
#4.100pg,>300bp	7.41ng/ul     74.1ng
  • Post-size amplification:
Double confirm dsDNA with selected size range
Produce enough DNA for DSN treatment
Option1: mix <300bp and >300bp )(10pg and 100pg) for DSN
Option2: reAmp for a few cycles individually
Take option 2, reAmp #1 and #2 for 3 cycles (goes with 4 cycles for safe), #3 and #4 for 1 cycle in 100ul PCR

File:12.18.12 reAmp.1-2.jpgFile:12.18.12 reAmp gel.jpg

  • beads purification, Qubit measurement
#1.10pg,<300bp	        13.3ng/ul       133ng
#2.10pg,>300bp	        13.1ng/ul       131ng	
#3.100pg,<300bp	4.65ng/ul(?)    93ng(?)
#4.100pg,>300bp	8.82ng/ul       176ng

DSN treatment[edit]

  • Reference: lab note on 11.7.12 [1]
  • it seems after 1.5hr treatment, all smears shift down below 400bp. Therefore, for the >300 fraction, I decided to do 1hr DSN treatment; 2.5hrs for <300 fraction.
  • DSN treatment had been used for three times: 10.18.12 (flat after 1hr); 11.6.12 (reasonable delay); 11.17.12 (no much delay)
  • However, in term of protocol, 10.18.12 is the right one; 11.6.12 has two major issues - [NaCl], 2X instead of 10X DSN buffer was used due to problem in 10X. All of them leads to greatly reduced (10X more) DSN efficiency...
  • Anyway, decide to follow the 10.18.12's procedure.

Test[edit]

File:12.17.12 preDSN.jpgFile:12.17.12 postDSN.jpg

File:12.17.12 DSN gel.jpg

postDSN amplification[edit]

File:12.17.12 postDSN all.jpgFile:12.17.12 postDSN gel.jpg

  • Beads purification, 0.8X
  • elute in 11ul, 1ul for gel

Sequencing libraries[edit]

  • RL-totoRNA-DSN-Dec20: mix #1-#4 as listed volume below
  • Gel quantification
   series size concentation    Miseq   GAII_HL140
#1 10pg   <300  18.77ng/ul	5.33ul  2.66ul
#2 10pg   >300  18.84ng/ul	5.31ul  2.65ul
#3 100pg  <300  31.07ng/ul	3.22ul  1.67ul
#4 100pg  >300  43.98ng/ul	2.27ul  1.17ul
  • Miseq; GAII HL140,s2,PE100
  • Read 1: totoRNA read1.v2; Read 2: ILMN Read2; N2 Read