title: GSM9115307: ILC2s, MCAO, blood, 1d, rep6; Mus musculus; RNA-Seq
library_name: GSM9115307
library_construction_protocol: ILC2s from young (8-10 week-old) transgenic and wild-type male mice on a B6.Cg-Tg(CAG-GFP)Smoc were collected for the experiment. First, IL-33 was intraperitoneally injected into mice every 2 days for 7 days for in vivo expansion of ILC2s. Next, ILC2s were collected from the bone marrow and spleen of the mice. Cells negativbe for lineage markers (CD11b, Gr1, CD3ε, CD45R/B220, and Ter119) and positive for CD25 were sorted using MACS. After in vitro culturing for 14 days in the presence of IL-2, IL-7, IL-15 and IL-33, the cells were further sorted by FACS as CD45+Lin-ST2+KLRG1+CD127+ cells. To compare the transcriptional profiles of ILC2s at key time points, we intravenously transferred the isolated ILC2s 6 hours after MCAO and collected them at different time points. We collected the transferred ILC2s from the blood at day 1 (bld_1d, n=6) and day 5 (bld_5d, n=6), also from the brain at day 5 after MCAO (br_5d, n=6) for RNA-seq. In addition, ILC2s directly from culture medium (base, n=5) were also included as a baseline control. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the secondstrand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. After the library QC, it is pooled according to the effective concentrationand the target data output required for the experiment, then subjected to Illumina sequencing, which produces 150 bp paired-end reads. The basic principle of the sequencing is "Sequencing by Synthesis," where fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencingcluster extends its complementary strand, the addition of each fluorescently labeled dNTP releases a corresponding fluorescence signal. The sequencer captures these fluorescence signals and converts them into sequencing peaks through computer software, thereby obtaining the sequence information of the target fragment.
identifiers.namespace: GEO
identifiers.id: GSM9115307_r1