ERC000011
ENA-CHECKLIST
2022-08-22
ENA-FIRST-PUBLIC
2022-08-22
ENA-LAST-UPDATE
SAMEA110646187
External Id
University of Zurich, Institut of Anatomy
INSDC center alias
University of Zurich, Institut of Anatomy
INSDC center name
2022-08-22T00:13:43Z
INSDC first public
2022-08-22T00:13:43Z
INSDC last update
public
INSDC status
ERS12743894
SRA accession
E-MTAB-12082:HIF1_KO3
Submitter Id
10
week
age
ArrayExpress
broker name
natural killer cell
cell_type
house mouse
common name
Protocols: Splenic natural killer cells were isolated using EasySep™ Mouse NK Cell Isolation Kit (Stemcell). NK cells from two to three spleens (same genotype, same sex) were pooled for each sample. General procedure: The tissue samples were homogenized in RLT buffer (Quiagen). Total RNA was isolated with Qiagen RNA extraction kits (Qiagen, 74104) following the manufacturer’s instructions. RNAseq from total RNA was performed by Novogene Europe. A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo- attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
description
adult
developmental stage
(NCR1-Cre) x C57BL/6-Hif1a(loxP)
genotype
SK5913, SK5913
individual
Mus musculus
http://purl.obolibrary.org/obo/NCBITaxon_10090
organism
Mus musculus
organism
spleen
organism part
Hif1a knockout
phenotype
male
sex
C57BL/6
strain
HIF1_KO3
title