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Jie:LabNotes/CpgSeq/2009-7-2
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==DNA and RNA extraction from samples and ASE validation for fuzzlmethylated genes== ===DNA and RNA extraction with Qiagen Allprep DNA/RNA Mini kit=== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|'''sample ''' |align="center" style="background:#f0f0f0;"|'''DNA concentration''' | align="center" style="background:#f0f0f0;"|'''DNA 260/280;260/230''' | align="center" style="background:#f0f0f0;"|'''RNA concentration''' | align="center" style="background:#f0f0f0;"|'''RNA 260/280;260/230''' |- |GM12864||94.1ng/ul x 100ul||1.97/0.17||1515.7ng/ul x 30ul||2.1/2.2 |- |GM12865||130.9ng/ul/ul x 100ul||1.98/1.44||1515.9ng/ul x 30ul||2.11/2.19 |- |GM12872||217.8ng/ul x 100ul||1.94/1.83||1638ng/ul x 30ul||2.1/2.18 |- |GM12873||82.6ng/ul x 100ul||1.92/1.49||1416.4ng/ul x 30ul||2.11/2.14 |- |GM12874||189.5ng/ul x 100ul||1.92/1.04||1829.4ng/ul x 30ul||2.11/2.14(performed on Jul 10th) |- |GM12875||140ng/ul x 100ul||1.89/1.03||838.3ng/ul x 30ul||2.13/2.11 |- |} ===Making cDNA === 64 65 72 73 75 74 RNA 3ul 3ul 3ul 3ul 4ul 3ul 10X DNase I buffer 5ul 5ul 5ul 5ul 5ul 5ul RNase-Free DNase I 2.5ul 2.5ul 2.5ul 2.5 2.5 2.5 H2O 39.5ul 39.5ul 32.5ul 32.5 31.5 32.5 37C 15min Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit * Add 4 volumes of RNA binding buffer; * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; * Add 200ul RNA Wash buffer, spin @15k rpm for 30s; * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; Reverse transcription 64~75 RNA 8ul 10mM dNTP 1ul 50uM dT12-18 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 25mM MgCl2 4ul 0.1M DTT 2ul RNaseOUT 1ul Superscript III 1ul Incubate all tube at 50C for 50min. Cleanup the first-strand reactions with qiaquick columns. quantify: GM12864: 74.8ng/ul; 2.17/1.16; GM12865: 72.3ng/ul; 2.16/1.09; GM12872: 79.1ng/ul; 2.08/0.78; GM12873: 76.4ng/ul; 2.16/1.17; GM12875: 66.4ng/ul; 2.17/1.14; GM12874: 120.6ng/ul;2.14/1.70. ===PCR to validat the ASE=== The primers and PCR conditions had been optimized on 09-06-29. refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2009-6-29] x 40 2x iQ supermix 20 800 Primer F (25uM) 0.5 0.5 each Primer R (25uM) 0.5 0.5 each cDNA samples as above: 0.1 dH2O 20 800 total 40ul For 12864/12872/12873: A1, C1-E1, A3-C3, E3-F3, H3, A5. For 12865/12875: G1, H1, D3. The annealing temperature for A5 and H1 is 58C. Annealing Temperature for others are 62C. 96C 3min-> 45x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min. [[Image:20090704_ASE_valid_1.jpg]]20090704_ASE_valid_1 [[Image:20090704_ASE_valid_2.jpg]]20090704_ASE_valid_2 I repeated the amplification of B1, D1-H1, A3-B3, D3, H3 with 42 PCR cycles. [[Image:20090706_ASE_valid_3.jpg]]20090706_ASE_valid_3 [[Image:20090706_ASE_valid_4.jpg]]20090706_ASE_valid_4 I purified the amplicons and selected 37 amplicons from 14 genes to Genewiz to do the sanger sequencing on 20090709. The plate label: [[Media: 20090709_plate_to_Genewiz_for_ASE_valid.xls]]20090709_plate to Genewiz for ASE valid ===results of ASE sanger sequencing results=== I got high quality reads for 10 genes. ATF7IP, C20orf96, PAN2, PRIC285, DNMT3B, LSP1 are not imprinted genes. No ASE were demonstrated either. CPNE8: No65: T; No75 C/T. (I did not do No74 yet. The Hapmap database show that No74 should be CC. <BS>However, if it is imprinted gene, father No75 should demonstrate monoallelic expression as well. <BS>I need to do the No74 ASE) COX412: need resequence using forward primer; GGT7: need to redo No64. GLANT9: need to redo No65,74,75. GLIPR1L2: No64 is C, No 72 is T, No 73 is C. This is a imprinted gene candidate. <BS>I need to repeat the No64 and do DNA sequencing validation. KIAA1853: need to purify the amplicon and redo the sequencing. MATN4: need to redo the No65, 74,75 ASE. c20orf85: need to redo the No64, 72, 73 ASE. ===redo the ASE PCR=== No74: CPNE8 G1 62C annealing 45cycles GLANT9 H1 58C MATN4 D3 62C GLIPR1L2 B3 62C No65: GLANT9 H1 MATN4 D3 GLIPR1L2 B3 62C No75: GLANT9 H1 MATN4 D3 GLIPR1L2 B3 62C No64: GGT7 A3 62C GLIPR1L2 B3 64C C20orf85 B1 58C No72: C20orf85 B1 No73: C20orf85 B1 [[Image:20090711_ASE_redo.jpg]]20090711_ASE_redo I sent the 22 samples to do the sanger sequencing on 09-07-13. [[Media:20090713_plate_to_genewiz_ASE_valid.xls|20090713_plate_to_genewiz_ASE_valid]] I got the results validated the monoallele expression of GLIPR1L2 of GM10864. I got the results validated the monoallelic expression of GLIPR1L2 on GM10865, 74 and 75 family. <BR>However, this gene is not imprinted gene. It demonstrated ASE. I designed the primer to validate the ASM of GLIRP1L2. [[Media:20090719_primer_GLIPR1L2_ASM_valid.xls|20090719_primer_GLIPR1L2_ASM_valid]] [[Media:20090719_GLIPR1L2_ASM_gDNA_target_seq.txt|20090719_GLIPR1L2_ASM_gDNA_target_seq]] I did not get the specific amplification of MATN4 and GLANT9. ===ASM validation of GLIPR1L2 on BJ(51ng/ul)refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-8-4] and BJ-iPS11(65.6ng/ul) refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq2008-8-15] bisulfite DNA=== 2x iQ supermix 20 Primer F (100uM) 0.15 Primer R (100uM) 0.15 bisulfite DNA: 0.1 dH2O 20 total 40ul 96C 3min-> 40x (95C 30sec, 62C 1min, 72C 1min)-> 72C 5min. I got the amplification from BJ-iPS11 only. [[Image:20090721_ASM_GLIPR1L2.jpg]]20090721_ASM_GLIPR1L2 I did the egel purification and clone the amplicon into the vector.
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