|
| Status |
Public on Mar 30, 2021 |
| Title |
untreated replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
iPSC-derived macrophages
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC-derived macrophages
treatment: untreated
|
| Treatment protocol |
Cells were treated with respective antibody at a concentration of 5 ug/ml or left untreated for 4 hours.
|
| Growth protocol |
Growth protocol according to Gutbier et al. 2020 (PMID: 32645954).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After treatment, cells were lysed in MagNA Pure LC RNA Buffer (Roche Diagnostics) and purified using the MagNA Pure 96 System (Roche Diagnostics).
10 ng of total RNA from each biological replicate were reverse transcribed to cDNA using Super Script IV Vilo (Thermo Fisher, 11766500, user guide MAN0015862). Libraries were generated with the AmpliSeq Library Plus Kit (Illumina Catalog number 20019103) according to the Reference Guide. Pipetting Steps for target amplification, primer digestion and adapter ligation were done with the mosquito automatic pipettor (ttp labtech) in a miniaturisation fashion. For the purifications before and after final library amplification, SPRI magnetic bead purification was chosen (Clean NGS, LABGENE Scientific SA) semi automated on multidrop (Thermo Fisher). Amplicon size and DNA concentration were measured using an Agilent High Sensitivity DNA Kit (Agilent Technologies, Waldbronn, Germany) according to the manufacturer’s recommendation. Prior sequencing samples have to be normalized and pooled to 2nM final concentration on Biomek FXP workstation.
Amplicon sequencing. Pooled libraries were sequenced on the Illumina NovaSeq 6000 Instrument, SBS (sequencing by synthesis) technology. 75 cycles end up with a minimum of 2 Mio SR per sample for analysing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Untreated.2
CD22_counts.gct
|
| Data processing |
Basecalling and sample de-multiplexing were performed with CASAVA-1.8.4 and default parameters.
Reads were quality check with fastqc v0.11.9 and multiqc v1.8.
Reads were mapped to the amplicon sequences of the panel using Bowtie2 v2.3.5 with the mapping parameters --no-mixed, --no-discordant and --sensitive
multiBamCov from bedtools v2.29.2 was used to count reads mapped onto amplicons.
Genome_build: Human RefSeq transcripts for amplicons.
Supplementary_files_format_and_content: CD22_counts.gct: GCT format, with amplicon counts for pathway reporter genes in each sample.
|
| |
|
| Submission date |
Mar 19, 2021 |
| Last update date |
Mar 30, 2021 |
| Contact name |
Roland Schmucki |
| E-mail(s) |
roland.schmucki@roche.com
|
| Organization name |
F. Hoffmann - La Roche AG
|
| Street address |
Grenzacherstrase
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE169252 |
CD22 blockage restores age-related impairments of microglia surveillance capacity |
|
| Relations |
| BioSample |
SAMN18384018 |
| SRA |
SRX10393037 |
