Homo sapiens
Protocols: Lymphoblast cell lines were established with EBV immortalization for three affected individuals (1-III-16, 1-IV-9, and 1-IV-11) and one healthy spouse (1-III-24) from Family 1, three affected individuals (5-III-2, 5-III-3 and 5-IV-4) from Family 5 and three healthy controls (Ctrl1-3). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples of donors using a standard ficoll procedure. B lymphocytes present in the culture were immortalized with Epstein-Barr virus in the presence of cyclosporin A. Not applicable Not applicable Not applicable Not applicable Total RNA including small RNAs were extracted from immortalized lymphoblastic cells from each individual using the AllPrepR DNA/RNA/miRNA kit (Qiagen). RNA-Seq libraries were generated from 600 ng of total RNA using TruSeq Stranded mRNA Sample Preparation Kit (Part Number RS-122-2101, Illumina). Briefly, following purification with poly-T oligo-attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 2 minutes. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second-strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double-stranded cDNA fragments, the products were purified and enriched with PCR (30 s at 98oC; [10 s at 98oC, 30 s at 60oC, 30 s at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman Coulter) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.
ENA-FIRST-PUBLIC
2019-09-16T04:03:09Z
ENA-LAST-UPDATE
2019-09-04T16:47:21Z
External Id
SAMEA5934373
INSDC center name
IGBMC
INSDC first public
2019-09-16T04:03:09Z
INSDC last update
2019-09-04T16:47:21Z
INSDC status
public
Submitter Id
E-MTAB-8301:CSDN4
broker name
ArrayExpress
cell type
B cell
clinical information
family 1
common name
human
developmental stage
adult
disease
Benign adult familial myoclonic epilepsy
individual
CSDN4
infect
Human gammaherpesvirus 4
organism part
blood
sample name
E-MTAB-8301:CSDN4
scientific_name
Homo sapiens