UTHSC CEPH B-cells Illumina (Sep09) RankInv

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Summary

The Illumina Human Whole Genome 6 v2.0 Rank Invariant data for CEPH lymphoblastoid cell lines obtained from the Coriell Institute for Medical Research. All cell lines were processed in Memphis in the UTHSC laboratory of Dr. Malak Kotb (2007-2009), by Dr. Rita G. Kasal, Nourtan Abdeltawab, and colleagues.

Selection of CEPH families and members was done by Dr. Roel Ophoff (Utrecht and UCLA).

Array data were generated at microarray core facility in laboratory of Dr. Weikuan Gu at VA medical center, Memphis, TN.

Analysis by Mark Reimers, Stephanie Santorico, and Roel Ophoff

Experiment design

Experimental Design and Batch Structure:

This data set consists of arrays processed in seven groups. Groups consisted of 2, 3, 4, 4, 3, 6, and then 8 beadchips at a time, batch IDs are indicated in table 1. Samples from same family were scattered among array groups, with samples from six different families were run on one chip. This was done to ensure balance and to minimize batch effects and group-by-family statistical confounds in normalization. This was done with the exception of the first two chips, which were run with 3 generations of the same family on one chip. A single operator, Yan Jiao, processed all arrays using illumina protocol for hybridization, washing and scanning. All samples in a group were labeled on one day, hybridization station accommodates up to 24 samples, or 4 beadchips. Chips were scanned using BeadArray Reader in sets of three.

About the processing of cell lines:

CEPH/UTAH families cell lines were purchased from Coriell repository of cell lines part of NIGMS. Upon arrival from the Coriell institute, we incubated the cell lines in 25ml flasks upright overnight at 37 ºC humidified incubator, with 5% carbon dioxide. We maintained the cells at a density of 5 X 105 cells/ml. The composition of the media used was RPMI-1640, 15% fetal bovine serum (FBS) and 2mM L-Glutamine; all FBS used was from the same lot. At 48 hours or when cell counts were ≥ 8 x 106 cells total, we harvested the cells and tested each cell line for mycoplasma contamination using e-Myco Mycoplasma PCR detection kit (iNtRON Biotechnology) according to manufacturer protocol. Cell lysates free of mycoplasma were used for RNA extraction as detailed below. We froze duplicates of each cell line at a concentration of ~2–6 x106 cells/ml according to standard procedures and stored in liquid nitrogen.

About RNA processing:

Two hundred and five cell lines were used for isolation of RNA. Ms. Sarah Rowe Hasty performed initial RNA isolation, purification and re-precipitation from 205 cell lines in Dr. Malak Kotb laboratory at VAMC. After initial RNA isolation, Ms. Nourtan Abdeltawab treated all samples for removal of contaminating DNA, along with further purification and re-precipitation of all samples. RNA samples that passed quality control were used to generate cRNA samples, those that didn't pass QC were re-extracted as we had duplicates of all cell lines lysates. RNA Extraction details: We used Qiagen RNeasy Mini purification of total RNA from tissues and cells spin protocol. RNA was isolated from 7.5 X 105 cells in duplicates. We froze cell lysates in RLT buffer and ß-mercaptoethanol at -80 ºC in 96 well plates until processed at a later time. We thawed samples, one 96 well plate at a time, and proceeded with RNA isolation steps and resuspended the pellets in RNase-free water. We then treated RNA to remove any DNA contamination using DNase digestion with RNase-free DNase kit (Qiagen) according to manufacturer protocol. RNA was finally purified by re-precipitation using ethanol precipitation using Purescript RNA purification kit (Gentra). Final purified RNA was resuspended in RNase-free water. RNA quality control: RNA samples were checked for RNA purity and integrity. RNA purity was evaluated using the 260/280 and 260/230 absorbance ratios. We used RNA samples with 260/280 ratio values ≥ 1.8 and 260/230 of ≥1.7. In cases were RNA samples did not meet these ratios, the RNA was purified by re-precipitation as above. RNA integrity was assessed using 1% RNA denaturing agrose gels. We required clear sharp bands of 18S and 28S rRNA for all samples compared to a control RNA sample to ensure intactness of rRNA.

All RNA samples were processed by Yan Jiao at Dr. Weikuan Gu’s microarray core facility at VA medical center, Memphis, TN. We used only RNA samples that passed quality control as detailed above and of concentration ≥ 50ng/ul for cRNA synthesis using Illumina TotalPrep RNA amplification kit (Ambion) according to manufacturer protocol. The basic outline of the procedure involves reverse transcription of RNA to synthesize cDNA using oligo (dT) primer, followed by in vitro transcription of purified dsDNA to synthesize amplified biotinylated cRNA (aRNA). We evaluated purified labeled cRNA using same methods as mentioned above for RNA samples. cRNA samples of good quality (passing QC), were then used to hybridize to Illumina Human-6WG v2.0 according to Illumina standard protocols.

About cases

About the CEPH/UTAH families used to generate this data set:

The CEPH/UTAH families used in this data set are part of CEPH repository linkage families of National Institute of General Medical Sciences (NIGMS) human genetic cell repository. These are immortalized human B-lymphocytes (EBV-transformed) from Caucasian donors of UTAH/Mormon ethnicity. The CEPH/UTAH families contain 48 families; the present data set includes 14 of these families with available DNA/genotypes for each member of these pedigrees. There are five families common with the published Monks et al (2004), namely families: 1346, 1362, 1418, 1421, and 1424.

Index Repository
Number		 Case ID Gender Family ID Family Member ID Sentrix ID Sentrix
Position		 Batch ID
1 GM07038 1333001 Male 1333 1 4256249060 A 5
2 GM06987 1333002 Female 1333 2 4158260019 B 4
3 GM07004 1333003 Male 1333 3 4256249103 A 7
4 GM07052 1333004 Male 1333 4 4256249051 A 6
5 GM06982 1333005 Male 1333 5 4158260009 F 3
6 GM07011 1333006 Female 1333 6 4158260012 C 3
7 GM07009 1333007 Male 1333 7 4256249101 F 6
8 GM07678 1333008 Male 1333 8 4158260005 B 4
9 GM07026 1333009 Male 1333 9 4158260026 C 3
10 GM07679 1333010 Male 1333 10 4158260030 C 3
11 GM07049 1333011 Male 1333 11 4256249059 A 5
12 GM07002 1333012 Female 1333 12 4158260001 B 4
13 GM07017 1333013 Male 1333 13 4158260027 B 5
14 GM07341 1333014 Female 1333 14 4256249042 A 6
15 GM11820 1333015 Female 1333 15 4256249104 E 7
16 GM07048 1341001 Male 1341 1 4256249103 B 7
17 GM06991 1341002 Female 1341 2 4158260027 A 5
18 GM07343 1341003 Female 1341 3 4256249095 C 7
19 GM07044 1341004 Female 1341 4 4158260002 C 4
20 GM07012 1341005 Female 1341 5 4158260026 D 3
21 GM07344 1341006 Female 1341 6 4158260030 B 3
22 GM07021 1341007 Male 1341 7 4256249097 B 7
23 GM07006 1341008 Female 1341 8 4158260001 C 4
24 GM07010 1341009 Female 1341 9 41582600011 A 2
25 GM07020 1341010 Male 1341 10 4158260007 A 1
26 GM07034 1341011 Male 1341 11 4158260007 B 1
27 GM07055 1341012 Female 1341 12 4158260012 E 3
28 GM06993 1341013 Male 1341 13 4256249059 C 5
29 GM06985 1341014 Female 1341 14 4158260007 C 1
30 GM10852 1346002 Female 1346 2 4256249098 D 7
31 GM12035 1346003 Male 1346 3 4158260007 D 1
32 GM12036 1346004 Male 1346 4 4256249047 A 6
33 GM12037 1346005 Male 1346 5 4158260027 E 5
34 GM12038 1346006 Male 1346 6 4158260001 E 4
35 GM12039 1346007 Male 1346 7 4256249097 C 7
36 GM12041 1346009 Female 1346 9 4256249099 B 7
37 GM12042 1346010 Female 1346 10 41582600011 D 2
38 GM12043 1346011 Male 1346 11 4158260007 E 1
39 GM12044 1346012 Female 1346 12 4256249096 F 7
40 GM12046 1346014 Female 1346 14 4158260007 F 1
41 GM10858 1347001 Male 1347 1 4158260030 A 3
42 GM10859 1347002 Female 1347 2 4158260027 C 5
43 GM11870 1347003 Female 1347 3 4158260018 C 2
44 GM11871 1347004 Male 1347 4 4256249060 B 5
45 GM11872 1347005 Male 1347 5 4256249097 A 7
46 GM11873 1347006 Male 1347 6 4158260001 A 4
47 GM11875 1347008 Female 1347 8 4256249095 E 7
48 GM11876 1347009 Male 1347 9 4256249059 E 5
49 GM11877 1347010 Male 1347 10 4158260021 A 2
50 GM11878 1347011 Male 1347 11 4158260008 A 1
51 GM11879 1347012 Male 1347 12 4158260019 D 4
52 GM11880 1347013 Female 1347 13 4158260008 B 1
53 GM11881 1347014 Male 1347 14 4256249101 C 6
54 GM11882 1347015 Female 1347 15 4158260008 C 1
55 GM11883 1347016 Male 1347 16 41582600011 B 2
56 GM10860 1362001 Male 1362 1 4256249097 E 7
57 GM10861 1362002 Female 1362 2 4158260027 D 5
58 GM11982 1362003 Female 1362 3 4158260019 F 4
59 GM11983 1362004 Female 1362 4 4158260030 D 3
60 GM11984 1362005 Male 1362 5 4158260008 D 1
61 GM11985 1362006 Female 1362 6 4158260002 F 4
62 GM11986 1362007 Female 1362 7 4256249060 C 5
63 GM11987 1362008 Male 1362 8 4158260005 F 4
64 GM11988 1362009 Female 1362 9 4256249042 B 6
65 GM11989 1362010 Female 1362 10 4158260001 F 4
66 GM11990 1362011 Male 1362 11 4256249094 A 6
67 GM11991 1362012 Female 1362 12 4158260018 E 2
68 GM11992 1362013 Male 1362 13 4256249059 B 5
69 GM11993 1362014 Female 1362 14 4256249059 F 5
70 GM11994 1362015 Male 1362 15 4158260008 E 1
71 GM11995 1362016 Female 1362 16 4158260008 F 1
72 GM11996 1362017 Male 1362 17 4158260021 C 2
73 GM10835 1416001 Male 1416 1 4256249060 F 5
74 GM10834 1416002 Female 1416 2 4158260018 A 2
75 GM12240 1416003 Male 1416 3 4158260019 E 4
76 GM12241 1416004 Male 1416 4 4256249059 D 5
77 GM12243 1416006 Male 1416 6 4256249096 C 7
78 GM12244 1416007 Female 1416 7 4256249047 B 6
79 GM12245 1416008 Male 1416 8 41582600011 E 2
80 GM12246 1416009 Female 1416 9 4158260026 E 3
81 GM12247 1416010 Female 1416 10 4158260021 D 2
82 GM12248 1416011 Male 1416 11 4256249094 B 6
83 GM12249 1416012 Female 1416 12 4256249041 A 6
84 GM12250 1416013 Male 1416 13 4256249044 F 7
85 GM12251 1416014 Female 1416 14 4158260005 D 4
86 GM12252 1416015 Female 1416 15 4158260009 E 3
87 GM12253 1416016 Female 1416 16 4158260027 F 5
88 GM10836 1418002 Female 1418 2 4158260018 B 2
89 GM12328 1418003 Female 1418 3 4256249047 C 6
90 GM12266 1418005 Female 1418 5 41582600011 F 2
91 GM12267 1418006 Male 1418 6 4158260009 D 3
92 GM12270 1418009 Female 1418 9 4158260001 D 4
93 GM12271 1418010 Female 1418 10 4158260012 D 3
94 GM12272 1418011 Male 1418 11 4256249060 E 5
95 GM12273 1418012 Female 1418 12 4158260002 D 4
96 GM12274 1418013 Male 1418 13 4256249101 A 6
97 GM12275 1418014 Female 1418 14 4158260005 C 4
98 GM10841 1421001 Male 1421 1 4158260030 E 3
99 GM10840 1421002 Female 1421 2 4256249098 C 7
100 GM12276 1421003 Female 1421 3 4158260012 F 3
101 GM12278 1421005 Female 1421 5 4256249042 C 6
102 GM12280 1421007 Female 1421 7 4256249041 B 6
103 GM12281 1421008 Male 1421 8 4256249047 D 6
104 GM12282 1421009 Male 1421 9 4158260026 F 3
105 GM12283 1421010 Female 1421 10 4256249101 B 6
106 GM12284 1421011 Male 1421 11 4256249094 C 6
107 GM12285 1421012 Male 1421 12 4256249104 B 7
108 GM12287 1421014 Female 1421 14 4256249051 B 6
109 GM11910 1423004 Female 1423 4 4256249103 D 7
110 GM11911 1423005 Female 1423 5 4256249096 E 7
111 GM11912 1423006 Male 1423 6 4158260009 C 3
112 GM11913 1423007 Male 1423 7 4256249044 D 7
113 GM11914 1423008 Female 1423 8 4256249098 A 7
114 GM11915 1423009 Female 1423 9 41582600011 C 2
115 GM11916 1423010 Male 1423 10 4158260018 D 2
116 GM11917 1423011 Male 1423 11 4256249097 F 7
117 GM11919 1423013 Male 1423 13 4256249104 F 7
118 GM11920 1423014 Female 1423 14 4256249095 D 7
119 GM11921 1423015 Female 1423 15 4256249099 A 7
120 GM10845 1424001 Male 1424 1 4256249104 C 7
121 GM10844 1424002 Female 1424 2 4256249103 F 7
122 GM11922 1424003 Male 1424 3 4256249044 A 7
123 GM11923 1424004 Male 1424 4 4256249097 D 7
124 GM11924 1424005 Female 1424 5 4256249096 A 7
125 GM11925 1424006 Male 1424 6 4158260021 B 2
126 GM11926 1424007 Female 1424 7 4256249051 C 6
127 GM11927 1424008 Male 1424 8 4256249042 D 6
128 GM11928 1424009 Male 1424 9 4256249098 B 7
129 GM11929 1424010 Male 1424 10 4256249094 D 6
130 GM11930 1424011 Male 1424 11 4256249041 C 6
131 GM11931 1424012 Female 1424 12 4158260002 A 4
132 GM11932 1424013 Male 1424 13 4256249095 A 7
133 GM11933 1424014 Female 1424 14 4158260005 E 4
134 GM12752 1447001 Male 1447 1 4158260021 E 2
135 GM12754 1447003 Female 1447 3 4256249044 E 7
136 GM12756 1447005 Male 1447 5 4256249098 F 7
137 GM12758 1447007 Female 1447 7 4256249099 D 7
138 GM12759 1447008 Female 1447 8 4158260002 E 4
139 GM12760 1447009 Male 1447 9 4256249103 E 7
140 GM12761 1447010 Female 1447 10 4158260009 A 3
141 GM12762 1447011 Male 1447 11 4158260018 F 2
142 GM12763 1447012 Female 1447 12 4158260012 A 3
143 GM12764 1447013 Male 1447 13 4256249042 E 6
144 GM12765 1447014 Male 1447 14 4158260026 A 3
145 GM12766 1451001 Male 1451 1 4158260009 B 3
146 GM12767 1451002 Female 1451 2 4158260012 B 3
147 GM12768 1451003 Male 1451 3 4256249041 D 6
148 GM12769 1451004 Female 1451 4 4158260021 F 2
149 GM12770 1451005 Female 1451 5 4158260005 A 4
150 GM12771 1451006 Male 1451 6 4256249047 E 6
151 GM12772 1451007 Female 1451 7 4158260019 A 4
152 GM12773 1451008 Female 1451 8 4158260002 B 4
153 GM12848 1451009 Female 1451 9 4256249051 D 6
154 GM12774 1451010 Male 1451 10 4158260030 F 3
155 GM12776 1451012 Female 1451 12 4256249099 E 7
156 GM12777 1451013 Male 1451 13 4256249060 D 5
157 GM12801 1454001 Male 1454 1 4256249103 C 7
158 GM12803 1454003 Female 1454 3 4158260019 C 4
159 GM12804 1454004 Female 1454 4 4256249101 D 6
160 GM12805 1454005 Male 1454 5 4256249095 F 7
161 GM12806 1454006 Male 1454 6 4256249098 E 7
162 GM12808 1454008 Female 1454 8 4256249099 C 7
163 GM12809 1454009 Female 1454 9 4256249051 E 6
164 GM12810 1454010 Male 1454 10 4256249104 A 7
165 GM12811 1454011 Male 1454 11 4256249044 B 7
166 GM12812 1454012 Male 1454 12 4256249042 F 6
167 GM12813 1454013 Female 1454 13 4256249094 E 6
168 GM12814 1454014 Male 1454 14 4256249096 D 7
169 GM12815 1454015 Female 1454 15 4256249041 E 6
170 GM12816 1454016 Male 1454 16 4256249047 F 6
171 GM12817 1456001 Male 1456 1 4256249051 F 6
172 GM12818 1456002 Female 1456 2 4256249101 E 6
173 GM12819 1456003 Male 1456 3 4256249095 B 7
174 GM12821 1456005 Female 1456 5 4256249044 C 7
175 GM12822 1456006 Male 1456 6 4256249094 F 6
176 GM12823 1456007 Male 1456 7 4256249096 B 7
177 GM12824 1456008 Female 1456 8 4256249104 D 7
178 GM12825 1456009 Male 1456 9 4158260026 B 3
179 GM12826 1456010 Female 1456 10 4256249041 F 6
180 GM12828 1456012 Female 1456 12 4256249099 F 7

About platform

Illumina HumanGW-6 v2.0 BeadChip: The Human-6 v2 beadchip simultaneously assays six samples, therefore, known as ‘array of arrays’. Each chip has ~1.8 million beads, beads have several hundred thousands copies of optimized 50-mer gene-specific probes. These probes cover more than 48,000 transcripts per sample, targeting genes and known alternative splice variants from the RefSeq database release 17 and UniGene build 188.

About data processing

About array data processing and analysis:

RNA samples (n = 180) were processed using a total of 30 Illumina HumanWG-6 BeadChips, each beadchip analyses six samples covering 48,000 transcripts per sample. All chips passed quality control and error checking. This data set was extracted and processed using the Bead Studio 3. We applied Rank-invariant normalization to all the samples and the resulting expression values along with gene ID were exported in GeneSpring format. Dr. Rita Kansal performed the normalization steps.

Contributors

Eldon Geisert, Barrett Haik, Malak Kotb, Lu Lu, Roel Ophoff, Robert Williams.

NIGMS Human Genetic Cell Repository

Acknowledgment

Financial support for this project was provided by Dr. Barrett Haik and the Hamilton Eye Institute, by NIH grant support to Malak Kotb, Robert W. Williams, Rita G. Kasal and colleagues, and by the UT Center for Integrative and Translational Genomics.