UTHSC CEPH B-cells Illumina (Sep09) RankInv (accession number: GN241) modify this page

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

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.

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.

About arrays used to generate this data set:

Array 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.

Sample Processing:

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.

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 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.

Financial support for this project was provided by Dr. Barrett Haik and the Hamilton Eye Institute, by NIH grant support to RWW and MK, and by the UT Center for Integrative and Translational Genomics.

Data entered by Arthur Centeno, Sept 22, 2009. This file started Sept 22, 2009 by RWW.

IndexRepository
Number
Case IDGenderFamily IDFamily Member IDSentrix IDSentrix
Position
Batch ID
1GM070381333001Male133314256249060A5
2GM069871333002Female133324158260019B4
3GM070041333003Male133334256249103A7
4GM070521333004Male133344256249051A6
5GM069821333005Male133354158260009F3
6GM070111333006Female133364158260012C3
7GM070091333007Male133374256249101F6
8GM076781333008Male133384158260005B4
9GM070261333009Male133394158260026C3
10GM076791333010Male1333104158260030C3
11GM070491333011Male1333114256249059A5
12GM070021333012Female1333124158260001B4
13GM070171333013Male1333134158260027B5
14GM073411333014Female1333144256249042A6
15GM118201333015Female1333154256249104E7
16GM070481341001Male134114256249103B7
17GM069911341002Female134124158260027A5
18GM073431341003Female134134256249095C7
19GM070441341004Female134144158260002C4
20GM070121341005Female134154158260026D3
21GM073441341006Female134164158260030B3
22GM070211341007Male134174256249097B7
23GM070061341008Female134184158260001C4
24GM070101341009Female1341941582600011A2
25GM070201341010Male1341104158260007A1
26GM070341341011Male1341114158260007B1
27GM070551341012Female1341124158260012E3
28GM069931341013Male1341134256249059C5
29GM069851341014Female1341144158260007C1
30GM108521346002Female134624256249098D7
31GM120351346003Male134634158260007D1
32GM120361346004Male134644256249047A6
33GM120371346005Male134654158260027E5
34GM120381346006Male134664158260001E4
35GM120391346007Male134674256249097C7
36GM120411346009Female134694256249099B7
37GM120421346010Female13461041582600011D2
38GM120431346011Male1346114158260007E1
39GM120441346012Female1346124256249096F7
40GM120461346014Female1346144158260007F1
41GM108581347001Male134714158260030A3
42GM108591347002Female134724158260027C5
43GM118701347003Female134734158260018C2
44GM118711347004Male134744256249060B5
45GM118721347005Male134754256249097A7
46GM118731347006Male134764158260001A4
47GM118751347008Female134784256249095E7
48GM118761347009Male134794256249059E5
49GM118771347010Male1347104158260021A2
50GM118781347011Male1347114158260008A1
51GM118791347012Male1347124158260019D4
52GM118801347013Female1347134158260008B1
53GM118811347014Male1347144256249101C6
54GM118821347015Female1347154158260008C1
55GM118831347016Male13471641582600011B2
56GM108601362001Male136214256249097E7
57GM108611362002Female136224158260027D5
58GM119821362003Female136234158260019F4
59GM119831362004Female136244158260030D3
60GM119841362005Male136254158260008D1
61GM119851362006Female136264158260002F4
62GM119861362007Female136274256249060C5
63GM119871362008Male136284158260005F4
64GM119881362009Female136294256249042B6
65GM119891362010Female1362104158260001F4
66GM119901362011Male1362114256249094A6
67GM119911362012Female1362124158260018E2
68GM119921362013Male1362134256249059B5
69GM119931362014Female1362144256249059F5
70GM119941362015Male1362154158260008E1
71GM119951362016Female1362164158260008F1
72GM119961362017Male1362174158260021C2
73GM108351416001Male141614256249060F5
74GM108341416002Female141624158260018A2
75GM122401416003Male141634158260019E4
76GM122411416004Male141644256249059D5
77GM122431416006Male141664256249096C7
78GM122441416007Female141674256249047B6
79GM122451416008Male1416841582600011E2
80GM122461416009Female141694158260026E3
81GM122471416010Female1416104158260021D2
82GM122481416011Male1416114256249094B6
83GM122491416012Female1416124256249041A6
84GM122501416013Male1416134256249044F7
85GM122511416014Female1416144158260005D4
86GM122521416015Female1416154158260009E3
87GM122531416016Female1416164158260027F5
88GM108361418002Female141824158260018B2
89GM123281418003Female141834256249047C6
90GM122661418005Female1418541582600011F2
91GM122671418006Male141864158260009D3
92GM122701418009Female141894158260001D4
93GM122711418010Female1418104158260012D3
94GM122721418011Male1418114256249060E5
95GM122731418012Female1418124158260002D4
96GM122741418013Male1418134256249101A6
97GM122751418014Female1418144158260005C4
98GM108411421001Male142114158260030E3
99GM108401421002Female142124256249098C7
100GM122761421003Female142134158260012F3
101GM122781421005Female142154256249042C6
102GM122801421007Female142174256249041B6
103GM122811421008Male142184256249047D6
104GM122821421009Male142194158260026F3
105GM122831421010Female1421104256249101B6
106GM122841421011Male1421114256249094C6
107GM122851421012Male1421124256249104B7
108GM122871421014Female1421144256249051B6
109GM119101423004Female142344256249103D7
110GM119111423005Female142354256249096E7
111GM119121423006Male142364158260009C3
112GM119131423007Male142374256249044D7
113GM119141423008Female142384256249098A7
114GM119151423009Female1423941582600011C2
115GM119161423010Male1423104158260018D2
116GM119171423011Male1423114256249097F7
117GM119191423013Male1423134256249104F7
118GM119201423014Female1423144256249095D7
119GM119211423015Female1423154256249099A7
120GM108451424001Male142414256249104C7
121GM108441424002Female142424256249103F7
122GM119221424003Male142434256249044A7
123GM119231424004Male142444256249097D7
124GM119241424005Female142454256249096A7
125GM119251424006Male142464158260021B2
126GM119261424007Female142474256249051C6
127GM119271424008Male142484256249042D6
128GM119281424009Male142494256249098B7
129GM119291424010Male1424104256249094D6
130GM119301424011Male1424114256249041C6
131GM119311424012Female1424124158260002A4
132GM119321424013Male1424134256249095A7
133GM119331424014Female1424144158260005E4
134GM127521447001Male144714158260021E2
135GM127541447003Female144734256249044E7
136GM127561447005Male144754256249098F7
137GM127581447007Female144774256249099D7
138GM127591447008Female144784158260002E4
139GM127601447009Male144794256249103E7
140GM127611447010Female1447104158260009A3
141GM127621447011Male1447114158260018F2
142GM127631447012Female1447124158260012A3
143GM127641447013Male1447134256249042E6
144GM127651447014Male1447144158260026A3
145GM127661451001Male145114158260009B3
146GM127671451002Female145124158260012B3
147GM127681451003Male145134256249041D6
148GM127691451004Female145144158260021F2
149GM127701451005Female145154158260005A4
150GM127711451006Male145164256249047E6
151GM127721451007Female145174158260019A4
152GM127731451008Female145184158260002B4
153GM128481451009Female145194256249051D6
154GM127741451010Male1451104158260030F3
155GM127761451012Female1451124256249099E7
156GM127771451013Male1451134256249060D5
157GM128011454001Male145414256249103C7
158GM128031454003Female145434158260019C4
159GM128041454004Female145444256249101D6
160GM128051454005Male145454256249095F7
161GM128061454006Male145464256249098E7
162GM128081454008Female145484256249099C7
163GM128091454009Female145494256249051E6
164GM128101454010Male1454104256249104A7
165GM128111454011Male1454114256249044B7
166GM128121454012Male1454124256249042F6
167GM128131454013Female1454134256249094E6
168GM128141454014Male1454144256249096D7
169GM128151454015Female1454154256249041E6
170GM128161454016Male1454164256249047F6
171GM128171456001Male145614256249051F6
172GM128181456002Female145624256249101E6
173GM128191456003Male145634256249095B7
174GM128211456005Female145654256249044C7
175GM128221456006Male145664256249094F6
176GM128231456007Male145674256249096B7
177GM128241456008Female145684256249104D7
178GM128251456009Male145694158260026B3
179GM128261456010Female1456104256249041F6
180GM128281456012Female1456124256249099F7