UTHSC CEPH B-cells Illumina (Sep09) RankInv

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

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.

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