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Sample GSM7328891

Query DataSets for GSM7328891

Status

Public on Aug 01, 2023

Title

Rat CD4+ T-MBP cells with validation library, spleen, rep R1

Sample type

SRA

Source name

Sorted CD4+ T-MBP cells from spleen

Organism

Rattus norvegicus

Characteristics

cell type: Sorted CD4+ T-MBP cells from spleen
genotype: Validation screen KO
strain: Lewis rat
time: day 3 post transfer

Growth protocol

Cells were transferred to Lewis rats three days before isolation. Prior to transfer, cells were kept in TCGF (complete DMEM supplemented with 10 % Horse serum and 2 % of PMA-stimulated EL4IL2 cell culture supernatant and 1 % Pen/Strep) for four days to expand the number of T cells. Then, the T cells were restimulated for two days with 50 Gy irradiated thymocytes in complete DMEM supplemented with 1 % rat serum and 10 µg/ml MBP, and the library was transduced by retroviral infection ontp Nycoprep purified T-MBP cells. For four days after transduction, T cells were kept in 0.5 ug/ml puromycin to select for infected cells. Cells were restimulated and transferred on the second day after restimulation

Extracted molecule

genomic DNA

Extraction protocol

The spleen was dissected and homogenized by passing through a metal strainer. Erythrocytes were removed by treating with ACK buffer for 3 min on ice and CD4+ T cells were enriched using the EasySep™ Rat CD4+ T Cell Isolation Kit (StemCell technologies). BFP+ Tcells were FACS sorted
Genomic DNA (gDNA) from lymphocytes or sorted TMBP cells was isolated with the DNeasy Blood and Tissue Kit (Qiagen). A one-step PCR amplification was performed with Q5 High Fidelity DNA Polymerase by using 2.5µg of gDNA per reaction with Fwd-Lib (mix of 8 staggered primers) and Rev-Lib (consists of 8bp of unique barcode) primers for a total of 24 cycles. Illumina adapters were introduced together with the amplification primers. All primer sequences are listed in Supplementary Table 1. The amplified DNA amplicons were purified with SPRIselect (Beckman Coulter) with a ratio of 1:0.8 (DNA to beads) and eluted in nuclease-free water. The presence of ~250bp DNA amplicons was confirmed and the concentration was measured with Agilent Bioanalyzer on DNA 1000 Chips (5067-1504).

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 1500

Description

Column names: R2Blood, R3Blood, R2Meninges, R3Meninges, R2Parenchyma, R3Parenchyma, R2Spleen, R3Spleen, R1Blood, R1Meninges, R1Parenchyma, R1Spleen

Data processing

The Galaxy platform was used for data analysis. For raw fastq files, Je-Demultiplex-Illu was used for de-multiplexing, followed by Cutadapt and Trimmomatic to get the 20bp sgRNA sequence.
Counts were then obtained with MAGeCK (version 0.5.7.1+) count. Normalization across samples was conducted in R (version 4.0.0+) after a 50 raw count threshold using the geometric mean per sgRNA for normalization, and sgRNAs with fewer than 50 counts in more than two replicates of the same tissue were discarded altogether.
The MAGeCK test was run without normalization or zero removal, and otherwise default parameters on Galaxy, giving the information of control Non-Targeting sgRNA for noise correction.
All further data processing was done with R.
Tab-delimited txt file of geometric mean R normalized sgRNA counts
Tab-delimited txt file of raw sgRNA counts from MAGeCK counts

Submission date

May 11, 2023

Last update date

Aug 01, 2023

Contact name

Martin Kerschensteiner

E-mail(s)

martin.kerschensteiner@med.uni-muenchen.de

Organization name

Institute for Clinical Neuroimmunology, Biomedical Center, University Hospital of the Ludwig-Maximilians University Munich

Street address

Großhaderner Str. 9