|
| Status |
Public on Nov 18, 2021 |
| Title |
N0972: BALBc Liver digested NKT1 CD4+ 23.000 cells |
| Sample type |
SRA |
| |
|
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CD4i+iNKT1 cells were lysed immediately following flow cytometry sorts, RNA was prepared using RNeasy Plus Mini Kit (QIAGEN)
1-5ng of total RNA were used for library preparation with the ‘SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian’ (#634413; Takara/Clontech) according to conditions recommended in user manual #063017. Generated libraries were barcoded by dual indexing approach and were finally amplified by 11 cycles of pcr. Fragment length distribution of generated libraries was monitored using ‘Bioanalyzer High Sensitivity DNA Assay’ (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the ‘Qubit® dsDNA HS Assay Kit’ (Q32854; ThermoFisher Scientific). Equal molar amounts of twelve to thirteen individually barcoded libraries were pooled for one sequencing run. Accordingly, each analyzed library constitutes 8.3% (7.7%) of overall flowcell capacity. The library pool was denatured with NaOH and was finally diluted to 1.5pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). 1.3 ml of denatured pool was loaded on an Illumina NextSeq 550 sequencer using a High Output Flowcell for 75bp single reads (#FC-404-2005; Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
BCL files were converted to FASTQ files using bcl2fastq Conversion Software version v2.20.0.422 (Illumina).
Raw data processing was conducted by use of nfcore/rnaseq (version 1.5dev). Sequenced reads were trimmed for adaptor sequence (Trim Galore! 0.4.4 dev), the reads were aligned (STAR2.5.3a_modified) and RNA reads were assigned and counted (featureCounts1.6.1). MultiQC report was generated with MultiQCv1.5.
Normalization and differential expression analysis was performed with DESeq2 (Galaxy Tool Version 2.11.40.2) with default settings except for “Output normalized counts table” which was set to “Yes”.
Genome_build: The genome reference and annotation data were taken from GENCODE.org (Mus musculus; GRCm38.p6; release M17).
Supplementary_files_format_and_content: DESeq2 was used to generate normalized read counts for all twenty samples. Statistics were generated by testing three contained Thymus samples against all other samples.
|
| |
|
| Submission date |
Oct 26, 2020 |
| Last update date |
Nov 18, 2021 |
| Contact name |
Hristo Georgiev |
| E-mail(s) |
Georgiev.Hristo@mh-hannover.de
|
| Phone |
05115329731
|
| Organization name |
Hannover Medical School
|
| Department |
Institute of Immunology
|
| Street address |
Carl Neuberg Str. 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21626 |
| Series (1) |
| GSE160144 |
Exploring the organ specific expression pattern of CD4+iNKT1 cells of C57BL/6 and BALB/c mice. |
|
| Relations |
| BioSample |
SAMN16555844 |
| SRA |
SRX9365393 |
