Rui:LabNotes/SingleCell/2012-12-17
Jump to navigation
Jump to search
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
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
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