Homo sapiens
Protocols: A skin biopsy was obtained from the P1 and P2 probands and four controls (C1-4). Primary fibroblasts were isolated and cultured. hTERT-RPE1 cells were maintained as described previously for the primary fibroblasts, but in DMEM/F-12, supplemented with 10% Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin. snRNA from P1, P2 and control fibroblasts were lysed using TRIzol and extracted with the Direct-zol RNA MiniPrep Plus (Qiagen and Zymo Research, respectively).   RNA in the snRNA size range (100-300 nt) was isolated from total RNA on 10% denaturing urea polyacrylamide gels; 4 µg total RNA was heat denatured for 2 minutes at 80 °C followed by cooling on ice and gel migration. The 100-300 nt size range was cut out, the gel was crushed and RNA was eluted in 300 µL 0.3M NaCl during 2h at room temperature. Then, the RNA was ethanol precipitated and resuspended in 20 µL water. Proper size selection was checked on an Agilent Bioanalyzer chip. Then, 10 µL of the RNA was treated with antarctic phosphatase to dephosphorylate RNAs other than the capped snRNAs by incubating for 30 minutes at 37 °C and 2 minutes enzyme inactivation at 80 °C. RNA was purified using AMPure beads (2x volume) and resuspended in 10 µL water. Next, snRNA were decapped with the RppH enzyme in 1x Thermopol buffer (20 mM Tris pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton) for 1 hour at 37 °C, followed by 5 minutes incubation at 65 °C. The RNA was again purified using AMPure beads (2x volume) and resuspended in 10 µL water.  Then, 6µL RNA was used for NGS library preparation using the NEBNext small RNA preparation kit following the manufacturer's instructions and 12 cycles of PCR amplification. Libraries were purified with AMPure beads (1.3x volume), quality checked using the Agilent Bioanalyzer
ENA first public
2024-01-01
INSDC center name
GIMM
INSDC status
public
Submitter Id
E-MTAB-14501:P2
broker name
ArrayExpress
cell_line
primary fibroblasts
cell_type
fibroblast
cohort
patient
collection date
not collected
common name
human
disease
retinal dystrophy with neurological impairment
geographic location (country and/or sea)
not collected
isolate
not applicable
sample name
E-MTAB-14501:P2
scientific_name
Homo sapiens