Supplemental material
Supplemental methods
1. Genotyping
Microsatellite D12Mit8
PCR reaction:
100 ng genomic DNA was used as template to perform PCRs in 20 µl reaction volume in presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.05 mM of each dNTP, 0.25 µM of primers D12Mit8 F and D12Mit8 R (supplemental table S4), and 0.1 U Taq DNA polymerase.
PCR conditions:
The reaction was denatured at 95° C for 3 min, subsequently 30 cycles were performed (94° C 1 min, 51° C 20 sec, 72° C 20 sec) followed by a final extension at 72° C for 5 min.
Microsatellite D12Mit259
PCR reaction:
For D12Mit259, 100 ng genomic DNA was used as template to perform PCRs in 20 µl reaction volume in presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.05 mM of each dNTP, 0.25 µM ofprimers D12Mit256 F and D12Mit256 R (supplemental table S4), and 0.1 U Taq DNA polymerase.
PCR conditions:
The reaction was denatured at 94° C for 1 min, subsequently 30 cycles were performed (94° C 1 min, 54° C 1 min, 72° C 20 sec) followed by a final extension at 72° C for 2 min.
Genotyping at the SNP in the 3’UTR of Begain
PCR reaction:
Genotyping at a polymorphic site in Begain exon 7 was done by single nucleotide primer extension on genomic PCR products amplified in presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.05 mM of each dNTP, 0.25 µM of primers exon 7 F and exon 7 R III (supplemental table 4), and 0.1 U Taq DNA polymerase.
PCR conditions:
The PCR conditions were as following: 5 min 95° C, 30 cycles [1 min 95° C, 1 min 60° C, 30 sec 72° C], 5 min 72° C.
SNuPE reaction:
5 µl of purified PCR products were used as templates for allele-specific primer extension in the presence of 3.6 µM SNuPE primer (supplemental table S4) , 0.05 mM of each ddNTP, 0.15 U Thermo-Sequenase (Amersham) in reaction buffer supplied by the manufacturer.
SNuPE conditions:
After denaturation for 2 min at 96° C 50 cycles (96° C 15 sec, 37° C 30 sec, 52.5° C 2 min) were performed.
2. RT-PCRs
cDNA synthesis:
For the allele-specific expression analysis of Wdr25, RNA was DNAseI treated for 30 min at 37° C followed by a 15 min inactivation at 95° C. For other RT-PCRs this step was omitted. cDNA was synthesised by reverse transcribing 1.9 µg total RNA in a 20 µl reaction volume at 37° C in presence of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 100 ng random primers, 0.8 U RNAsin, 0.375 mM of each dNTP, 7 U M-MLV reverse transcriptase (Promega). Success of the cDNA synthesis was tested by amplification of b-actin or Polr2a/POLR2A.
PCR reaction:
The PCRs were performed in 20 µl reaction volume in presence of 67 mM Tris-HCl, pH 8.8, 16 mM (NH4)2SO4, 0.01% Tween-20, 2 mM MgCl2, 0.05 mM of each dNTP, 0.25 µM of each primer, and 1 U LunaTaq DNA polymerase (Bioline). For C14orf70 amplicons, 1.25 M betain was added to the PCR reaction mix. Primer sequences are listed in supplemental table S1.
PCR conditions:
The reaction was denatured at 94° C for 5 min, subsequently 35 PCR cycles were performed (30 sec 94° C, 30 sec annealing [ß-actin 62° C, Polr2a/POLR2A 60° C, Wdr25 60° C, Begain 60° C], 1 min 72° C, followed by a final extension at 72° C for 5 min). b-actin was amplified in 30 cycles.
3. Real-Time RT-PCRs
cDNA synthesis:
cDNA was synthesised by reverse transcribing 1.9 µg total RNA in a 20 µl reaction volume at 37° C in presence of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 100 ng random primers, 0.8 U RNAsin, 0.375 mM of each dNTP, 7 U M-MLV reverse transcriptase (Promega).
PCR reaction:
Begain Real-Time RT-PCRs were performed in a 20 µl reaction volume in the presence of 1 µl cDNA, 250 pmol of primers exon 6 F and exon 7 R (supplemental table 4) and 9 µl of Brilliant Sybr Green QPCR Master Mix (Stratagene) and amplified on a MX3000 PCR machine (Stratagene). Polr2a amplification was performed with primers Polr2a F and Polr2a R using the same reaction conditions. As standard 1 µl of 1:10, 1:100, 1:1000 and 1:10.000 dilutions of 1.9 µg cDNA from neonatal brain were used.
PCR conditions:
Three technical replicates per RNA for Begain and Polr2a were performed starting with an initial denaturation step for 10 min at 95° C followed by 40 cycles of 95° C 30 sec, 60° C 1 min, 72° C 20 sec.
4. RT-PCRs for detection of allele-specific Begain transcription
cDNA synthesis:
3 µg RNA were denatured in a boiling water bath and reverse transcribed in 20 µl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM primer Begain exon 7 R II, 0.5 µM primer b-actin R, 0.8 U RNAsin, 0.375 mM of each dNTP, 7 U M-MLV reverse transcriptase (Promega) in a 37°C waterbath for 2 h 30 min followed by 15 min heat inactivation at 70°C. Alternatively, the primer 7 R I was used to proof consistency of the RT reaction.
RT-PCR on exon 1A containing transcripts:
PCR reaction:
PCR products were amplified from 1.7 µl cDNA in 30 µl volume in presence of 67 mM Tris-HCl, pH 8.8, 16 mM (NH4)2SO4, 0.01% Tween-20, 0.2 mM MgCl2, 0.17 mM of each dNTP, 1.17 M betain, 1.7% DMSO, 0.17 µM of exon 1A F and exon 7 R III RT-PCR primers (supplemental table S1) and 10 Units Taq DNA polymerase (invitrogen).
PCR conditions:
The PCR conditions were as following: 8 min 95° C, 35 cycles [2 min 95° C; 2 min 59.5° C; 3 min 30 s 72° C], 15 min 72° C.
RT-PCR on exon 1B containing transcripts
PCR reaction:
Transcripts starting with exon 1B were amplified from 1.7 µl cDNA in presence of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.17 mM of each dNTP, 1.17 M betain, 1.7% DMSO, 0.17 µM of exon 1B F and exon 7 R III RT-PCR primers (supplemental table S1), and 10 U Platinum Taq DNA polymerase (invitrogen).
PCR conditions:
The PCR conditions were as following: 8 min 95° C, 35 cycles [2 min 95° C; 2 min 59° C; 3 min 30 s 72° C], 15 min 72° C.
Nested-PCR on PCR products containing exon 1A or exon 1B
PCR reaction:
1 µl of purified PCR product was used as template for 17 cycles of nested PCR in presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.05 mM of each dNTP, 0.25 µM of primers exon 7 F and exon 7 R III (supplemental table 1), and 0.1 U Taq DNA polymerase.
PCR conditions:
The PCR conditions were as following: 5 min 95° C, 17 cycles [1 min 95° C, 1 min 60° C, 30 sec 72° C], 5 min 72° C.
5. Single nucleotide primer extension on Begain and Wdr25 RT-PCR products and HPLC separation
SNuPE reaction:
Approx. 100 ng of purified PCR product were used as templates for allele-specific primer extension in the presence of 3.6 µM Begain or Wdr25 SNuPE primers (supplemental table S1) , 0.05 mM of each ddNTP, 0.15 U Thermo-Sequenase (Amersham) in reaction buffer supplied by the manufacturer.
SNuPE conditions:
After denaturation for 2 min at 96° C 50 cycles (96° C 15 sec, 37° C 30 sec, 52.5° C 2 min) were performed.
HPLC separation:
Extension products were separated on a DHPLC system (WAVETM system, Transgenomic) at 50° C applying an acetonitrile gradient by continuously mixing buffer A (0.1 M triethylammonium acetate (TEAA)) and buffer B (0.1 M TEAA and 25% acetonitrile): Begain 20-32% buffer B for 15 min, Wdr25 19-30% buffer B for 12 min.
After estimation of the peak heights allele-specific expression levels were calculated as ratio of allele-specific expression to total expression of both alleles.
6. DNA methylation analysis by Southern blot hybridisation
Amplification and labelling of the probe
PCR reaction:
100 ng genomic DNA was used as template to perform PCRs in 20 µl reaction volume in presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.05 mM of each dNTP, 0.25 µM of primers probe exon 1B F and probe exon 1B R (supplemental table S1), 1.25 M Betain, and 0.1 U Taq DNA polymerase.
PCR conditions:
The reaction was denatured at 95° C for 3 min, subsequently 30 cycles were performed (94° C 30 sec, 60° C 45 sec, 72° C 30 sec) followed by a final extension at 72° C for 5 min.
Labelling:
The probe was labelled with the Megaprime DNA labelling system (Amersham Biosciences) and added to the ULTRAhyb (Ambion) hybridisation buffer.
Supplemental tables
Table S1: Primer sequences used in microsatellite PCR, RT-PCR, Real-Time PCR and SNuPE experiments
Table S2: Highly conserved elements between Wars and Dlk1
Highly conserved elements were selected from the alignment of the mouse and human genomic sequences generated using the Pipmaker software (http://bio.cse.psu.edu/cgibin/ pipmaker; Schwartz et al., 2000). The selected elements were at least 100 bp long and identical in at least 70% of the positions. Conserved elements overlapping with EST´s are highlighted in grey.
position in murineBAC clones / position in
human
BAC clones / length / identity / gene
AC122811.4 / AL157871.5
44679-44853 / 142837-143011 / 175 nt / 86% /
Wars
46218-46376 / 145025-145183 / 159 nt / 86%49431-49613 / 150364-150546 / 183 nt / 93%
51087-51242 / 154704-154859 / 156 nt / 80%
58530-58725 / 161651-161846 / 196 nt / 85%
59085-59295 / 162414-162624 / 211 nt / 79%
64461-64585 / 168520-168644 / 125 nt / 90%
66116-66337 / 169677-169898 / 222 nt / 83%
AC122811.4 / AL135838.6
81356-82156 / 72174-72977 / 801 nt / 75% /
Wdr25
87460-87570 / 68516-68626 / 111 nt / 79%87670-87814 / 68266-68410 / 144 nt / 90%
89685-90091 / 65655-66060 / 406 nt / 88%
90636-90864 / 64827-65055 / 229 nt / 88%
92443-92649 / 62534-62740 / 207 nt / 80%
94343-94551 / 60720-60928 / 209 nt / 91%
94593-94726 / 60539-60671 / 133 nt / 83%
96883-97003 / 57491-57611 / 121 nt / 84%
97479-97665 / 56660-56846 / 187 nt / 76%
99514-99618 / 54267-54371 / 105 nt / 87%
99640-99750 / 54141-54251 / 111 nt / 78%
100274-101058 / 52747-53534 / 785 nt / 91%
101802-102035 / 51705-51937 / 233 nt / 92%
106749-106942 / 50094-50287 / 194 nt / 83%
110472-110600 / 45002-45130 / 129 nt / 82%
111168-111560 / 44002-44395 / 393 nt / 90%
120391-120553 / 30910-31072 / 161 nt / 87%
121498-121571 / 30168-30270 / 102 nt / 70%
123558-123809 / 27483-27734 / 252 nt / 79%
128108-128298 / 22714-22905 / 191 nt / 74%
128421-128520 / 22485-22584 / 100 nt / 86%
128941-129041 / 21480-21580 / 101 nt / 74%
135125-135353 / 15723-15951 / 229 nt / 86%
135413-135726 / 15351-15666 / 313 nt / 87%
136774-136941 / 14131-14298 / 168 nt / 71%
137774-137994 / 12929-13149 / 221 nt / 90%
142254-142417 / 10889-11052 / 164 nt / 76%
142510-142716 / 10581-10787 / 207 nt / 74%
142978-143154 / 10134-10310 / 177 nt / 86%
144308-144427 / 8520-8639 / 121 nt / 71%
146163-146387 / 6021-6245 / 225 nt / 82%
AC122811.4 / AL845552.2
160689-160910 / 9866-10086 / 221 nt / 83%
164108-164267 / 15599-15758 / 160 nt / 88%
165790-166090 / 19140-19434 / 295 nt / 92%
166155-166260 / 19496-19601 / 106 nt / 73%
168069-168323 / 23872-24127 / 255 nt / 86%
AC140111.3 / AL845552.2
70884-71074 / 31549-31739 / 191 nt / 82%
72593-72722 / 33796-33925 / 130 nt / 82%
76528-76671 / 38390-38533 / 144 nt / 85%
88801-88912 / 51462-51573 / 112 nt / 81%
98954-99067 / 68490-68606 / 113 nt / 87%
100888-100992 / 71608-71712 / 105 nt / 77%
103034-103291 / 73419-73676 / 258 nt / 80%
104513-104682 / 76649-76818 / 170 nt / 85%
105339-105574 / 77391-77626 / 236 nt / 85%
111082-112477 / 85545-86919 / 1395 nt / 84% /
Begain
113420-113545 / 88066-88191 / 126 nt / 95%115068-115215 / 91408-91555 / 148 nt / 84%
116955-117126 / 94082-94253 / 172 nt / 88%
119491-119640 / 97980-98129 / 150 nt / 91%
AC140111.3 / AL132711.5
149552-149673 / 9735-9856 / 122 nt / 75%
AC126269.3 / AL132711.5
81631-81737 / 50989-51095 / 107 nt / 73%
AC107681.15 / AL132711.5
161035-161213 / 80601-80779 / 179 nt / 88%
156358-156466 / 86430-86538 / 109 nt / 82%
156164-156281 / 86637-86754 / 118 nt / 83%
152907-153006 / 92700-92799 / 100 nt / 81%
152320-152446 / 93563-93689 / 127 nt / 82%
150361-150461 / 96086-96186 / 101 nt / 76%
143156-143277 / 107449-107570 / 122 nt / 76%
131240-131377 / 118822-118959 / 138 nt / 71%
130884-131014 / 119189-119319 / 131 nt / 74%
126643-126759 / 124579-124695 / 117 nt / 79%
126188-126074 / 125135-125249 / 115 nt / 76%
122128-122302 / 129812-129986 / 175 nt / 75%
119560-119691 / 133911-134041 / 131 nt / 74%
117713-117944 / 135495-135726 / 232 nt / 80%
114229-114336 / 138810-138917 / 108 nt / 75%
112864-112965 / 140300-140401 / 102 nt / 76%
110121-110345 / 142412-142636 / 225 nt / 85% /
Dlk1
108212-108369 / 144422-144579 / 157 nt / 72%106310-106456 / 146768-146914 / 147 nt / 82%
105628-105806 / 147510-147688 / 179 nt / 75%
104691-104847 / 148815-148971 / 157 nt / 89%
103489-104262 / 149628-150395 / 768 nt / 91%
Table S3: Distribution of repetitive elements within the analysed mouse and human sequences. The segment positions refer to the map in figure 1B.
segment / G+C content / SINEs / LINEs / LINE1 / LTRs / retroviral elements0-210 kb / 50.48% / 7.59% / 6.40% / 5.90% / 3.65% / 19.47%
210 – 540 kb / 42.25% / 2.53% / 53.80% / 53.76% / 18.05% / 76.63%
540 kb – end / 51.51% / 6.57% / 1.10% / 0.69% / 1.49% / 9.51%
mouse (total) / 46.18% / 4.75% / 31.40% / 31.17% / 11.23% / 49.27%
human (total) / 51.14% / 13.64% / 16.62% / 11.25% / 4.82% / 37.28%
0-260 kb / 49.69% / 15.07% / 20.01% / 13.92% / 1.64% / 39.28%
260-320 kb / 50.91% / 14.27% / 16.48% / 12.15% / 22.64% / 55.20%
320-end / 55.92% / 8.87% / 7.74% / 1.65% / 1.82% / 19.80%
Table S4: Positions of Begain exons in the murine BAC clone AC140111.3