Human
BioSampleModel
2022-08-11
ENA-FIRST-PUBLIC
2022-08-11
ENA-LAST-UPDATE
SAMN28889691
External Id
pda|tdoniger
INSDC center alias
pda|tdoniger
INSDC center name
2022-08-11T03:15:09Z
INSDC first public
2022-08-11T03:15:09Z
INSDC last update
public
INSDC status
SRS13321028
SRA accession
SLK_RMS
Submitter Id
not applicable
age
not applicable
biomaterial_provider
NCBI
broker name
Human renal cell carcinoma iSLK cells
cell_line
not applicable
cell_type
human
common name
Total RNA (5mg) was denatured at 90oC for 2 min in a thermocycler. Then, an equal volume of buffer (NaHCO3/Na2CO3, pH 9.9) was added, and RNA samples were incubated at 90oC for 20 minutes. The hydrolyzed RNA was then used for library preparation. Briefly, 800ng of the RNA was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and cleaned by Agencourt RNA clean XP beads (Beckman Coulter). The RNA was then ligated to 3' linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing DMSO, ATP, PEG 8000 and RNase inhibitor (NEB) for 1.5hr at 22oC. The ligated RNA was purified from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase enzyme (Agilent) at 55oC for 45 min. Next, the RNA was degraded using 2ml of 1M NaOH, and the cDNA was cleaned using Dynabeads® MyOne™ SILANE beads (Thermo Scientific). The cDNA was further ligated to 3' adapter using a high concentration T4 RNA Ligase 1 (NEB) overnight at 22oC and cleaned of excess adapter by using Dynabeads® MyOne™ SILANE beads (Thermo Scientific). The adapter ligated cDNA was PCR enriched using NEBNext® High-Fidelity (NEB) polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel (Invitrogen) and size selected for the range of 150-300 bp (containing ~30-180nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and sequenced in a Nextseq system (Illumina) in paired end mode (20-40 million reads for each sample).
description
not applicable
isolate
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
Uninfected
phenotype
Total RNA
sample_type
not applicable
sex
not applicable
tissue
Homo sapiens
title
Alkaline Hydrolysis for 20'
treatment