Human
BioSampleModel
2022-08-11
ENA-FIRST-PUBLIC
2022-08-11
ENA-LAST-UPDATE
SAMN28889689
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
SRS13321025
SRA accession
SLK_HydraPsi
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 treated with 50% hydrazine (Sigma) for 45 minutes on ice, and ethanol precipitated. The RNA pellet was then resuspended in 1M aniline (Sigma) (pH 4.5 adjusted using glacial acetic acid) until the white pellet was completely dissolved, boiled for 15 minutes at 60oC in the dark, and immediately placed on ice. The fragmented RNA was recovered by ethanol precipitation, and used for library preparation. To perform HydraPsiSeq, the fragmented RNA (~800ng) was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), cleaned by Agencourt RNA clean XP beads (Beckman Coulter) and ligated to a 3' linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing DMSO, ATP, PEG 8000, and RNase inhibitor (NEB). The ligated RNA was cleaned from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase (Agilent). The RNA was subsequently degraded using 2ml of 1 M NaOH, and the cDNA was cleaned using Dynabeads MyOne SILANE beads (Thermo Scientific). The cDNA was further ligated to a 3' adapter using a high concentration T4 RNA Ligase 1 (NEB) and cleaned of excess adapter 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 at the range of 150-300 bp (containing ~30-180 nt 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 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
Hydrazine 45'-Aniline 15'
treatment