 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 04, 2023 |
| Title |
M31 Amplicon-NGS on treated cells (post transplantation) |
| Sample type |
SRA |
| |
|
| Source name |
HSPCs from bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
tissue: HSPCs
sample type: Amplicons from gDNA
genotype: Jinx; C57BL/6J-Unc13dJinx/Mmucd
treatment: Cas9+sgRNAs u3 and d4-treated
|
| Treatment protocol |
Lineage-negative (Lin-) HSCs (Miltenyi Biotec, 130-090-858) from femur and tibia-derived BM were stimulated for 3h in StemSpan SFEM II medium (StemCell Technologies) supplemented with L-glutamine (20 mM, Gibco), mSCF, mTPO and hIGF2 (20 ng/ml each) plus hIGF2 and mIL-3 (10 ng/ml each, Peprotech) before nucleofection (Lonza 4D nucleofector, P4 kit, program CA137) with two times 3µg Cas9 (IDT) complexed at a 1:3 molar ratio with sgRNAs (BioLegio). After 20h of culture without mIL-3, 10E6 cells were infused via tail vein to recipients conditioned with 5 cycles of busulfan (20 mg/kg, Fresenius Kabi) over 5 consecutive days. Cells were harvested from peripheral blood through tail vain bleeding or extracted from the indicated source upon sacrifice of the mice.
|
| Growth protocol |
Cells were cultured at 37°C and 5% CO2. Lineage-negative (Lin-) HSCs (Miltenyi Biotec, 130-090-858) from femur and tibia-derived BM were stimulated for 3h in StemSpan SFEM II medium (StemCell Technologies) supplemented with L-glutamine (20 mM, Gibco), mSCF, mTPO and hIGF2 (20 ng/ml each) plus hIGF2 and mIL-3 (10 ng/ml each, Peprotech) before nucleofection. After 20h of culture without mIL-3, 10E6 cells were infused via tail vein to recipients conditioned with 5 cycles of busulfan (20 mg/kg, Fresenius Kabi) over 5 consecutive days. Animals were kept in a dedicated facility.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from cells using theNucleoSpin Tissue Mini kit for DNA from cells and tissue (Macherey-Nagel) according to the manufacturer’s instructions.
DNA library preparations, sequencing reactions, and initial bioinformatics analysis were conducted at AZENTA, Inc. (Leipzig, Germany). DNA Library Preparation, clustering, and sequencing reagents were used throughout the process using NEBNext Ultra DNA Library Prep kit following the manufacturer’s recommendations (Illumina, San Diego, CA, USA). End repaired adapters were ligated after adenylation of the 3’ends followed by enrichment by limited cycle PCR. DNA libraries were quantified using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and multiplexed in equal molar mass.
The pooled DNA libraries were loaded on the Illumina instrument according to manufacturer’s instructions. The samples were sequenced using a 2x250 paired-end (PE) configuration. Image analysis and base calling were conducted by the Illumina Control Software on the Illumina instrument.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
The raw Illumina reads were checked for adapters and quality via FastQC. The raw Illumina sequence reads were trimmed of their adapters using Trimmomatic v. 0.36. Raw sequence data (.bcl files) generated from Illumina MiSeq were converted into fastq files and de-multiplexed using Illumina bsl2fastq v. 2.17 program.
Indels were detected using CRISPResso2 (https://github.com/pinellolab/CRISPResso2)
Assembly: TACATGAACACCAACCTGGTCCAGGAGAACTTCAGCAGgtacccagcagcccccgatctgccctcggccgccatgtctccaaggctcacgagtaagtggcatgttgggattccctttggttgggattccctttggtgacttccttggaggatgtaaccagcttcggggacagggagctcactgcttcccttaaaggtccttttttcttctgcccagctctaagcacggcaagcctttccctgtatccctccaggcctgggcccatcattaatggcccctaactgcatctccttcctct
Supplementary files format and content: Excel file containing the indels In comparison to the reference amplicon
Library strategy: Amplicon-seq
|
| |
|
| Submission date |
Feb 17, 2023 |
| Last update date |
Jul 04, 2023 |
| Contact name |
Gianni Monaco |
| E-mail(s) |
mongianni1@gmail.com
|
| Organization name |
Freiburg Medical Center
|
| Street address |
Breisacher Straße
|
| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE225540 |
Gene Editing of Hematopoietic Stem Cells Restores T Cell Response in a Familial Hemophagocytic Lymphohistiocytosis Model [Amplicon] |
| GSE225541 |
Gene Editing of Hematopoietic Stem Cells Restores T Cell Response in a Familial Hemophagocytic Lymphohistiocytosis Model |
|
| Relations |
| BioSample |
SAMN33339704 |
| SRA |
SRX19402941 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7050012_M31_Allele_freq_table.xlsx |
254.6 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
