nebnext ultra ii directional rna library prep kit with poly a selection  (New England Biolabs)

Journal: bioRxiv

Article Title: Microglia Piezo1 aggravates cerebral ischemia and reperfusion injury by chemotactic recruitment of T lymphocytes

doi: 10.64898/2026.01.12.699156

Figure Lengend Snippet: A-C RT-PCR analysis of relative Piezo1 mRNA expression in primary microglia (A), astrocyte (B) and neuron (C) upon normoxia and OGD/R condition, with normoxia set to 1.0 ( n = 4 per group). D RT-PCR analysis of the relative Piezo1 mRNA expression in the MACS microglia isolated from mice subjected to sham operation or MCAO/R injury, with sham brain set to 1.0. E, F Western blot analysis of Piezo1 in MACS microglia isolated from mice subjected to sham operation or MCAO/R injury. A representative image was shown in E panel. Quantification of Piezo1 protein in indicated groups was shown in F panel by taking sham brain set to 1.0. G, H Representative fura-2 Ca 2+ imaging of primary cultured microglia upon Normoxia or OGD/R condition response to Yoda1 (G) and ATP (H). I Scatterplot of maximal fura-2 amplitude changes in response to Yoda1 or ATP in indicated groups. J Representative immunofluorescence images of Piezo1 with Iba1 in the peri-infarcted regions after MCAO/R. Scale bar, 50 µm. K, L Quantification of the percentage of Piezo1 + cells among Iba1 + cells in peri-infarcted regions (K), and infarct cores (L). Data are expressed as mean ± SD. One-way ANOVA with Dunnett’s post hoc test for A, B, C, K and L. Two tailed unpaired Student’s t test for I. Kruskal–Wallis test with Dunn’s post hoc test for D, F. ** P < 0.01, *** P < 0.001, n.s.: no significant.

Article Snippet: Paired-end sequencing libraries were performed using a mRNA NEBNext Poly selection Library Prep Kit (Illumina, Los Angeles, CA, USA). cDNA sequence was then conducted using the Illumina NovaSeq 6000 sequencing platform with multiplex paired-end 150 base pairs reads.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Western Blot, Imaging, Cell Culture, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Microglia Piezo1 aggravates cerebral ischemia and reperfusion injury by chemotactic recruitment of T lymphocytes

doi: 10.64898/2026.01.12.699156

Figure Lengend Snippet: A,B Heatmap plot of multiplexed immunoassay showing chemokine expression profiles in the ischemic brains of indicated groups. Quantification of chemokine concentrations in indicated groups is shown in panel B, results were generated according to clustering of proteome profiler array assays of the listed chemokine. C Cxcl10 mRNA levels detected by RT-PCR in peri-infarct area of Piezo1 CKO mice within the first 72 h after MCAO/R. D.E Western blot analysis of CXCL10 in peri-infarct area separated from control and Piezo1 CKO mice subjected to sham operation or MCAO/R injury. Reprehensive image was shown in panel D. Quantification of CXCL10 protein in indicated groups was shown in panel E, with sham brain set to 1.0. F Immunostaining for CXCL10 and GFAP of brain sections 72 h after MCAO/R. Scale bar, 50 µm. G Quantitative analysis of the GFAP and CXCL10 positive cells number in panel F. H Representative Evans blue staining images of brain sections. I Quantitative analysis of the Evans blue staining volume in panel H. Each symbol represents one mouse. Data are expressed as mean ± SD. Two-tailed unpaired t-test for B and G. Two-way ANOVA followed by Sidak’s post hoc test for C and E. One-way ANOVA followed by Dunnett’s post hoc test for I. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: no significant.

Article Snippet: Paired-end sequencing libraries were performed using a mRNA NEBNext Poly selection Library Prep Kit (Illumina, Los Angeles, CA, USA). cDNA sequence was then conducted using the Illumina NovaSeq 6000 sequencing platform with multiplex paired-end 150 base pairs reads.

Techniques: Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Immunostaining, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Microglia Piezo1 aggravates cerebral ischemia and reperfusion injury by chemotactic recruitment of T lymphocytes

doi: 10.64898/2026.01.12.699156

Figure Lengend Snippet: A Relative cytokine mRNA levels in the peri-infarct area of control and Piezo1 CKO mice were detected by RT-PCR analysis 72 h after MCAO/R. B TNF-α in in peri-infarct area of control and Piezo1 CKO mice were detected by western blot analysis 72 h after MCAO/R. C Quantification of TNF-α of indicated groups, normalized to controls (set as 1.0). D flow cytometry analysis of IFN-γ positive cells in peri-infarct area of control and Piezo1 CKO mice 72 h after MCAO/R. E Quantitative analysis of IFN-γ positive cells in panel D. F, G Tnf-α (F) and Ifn-γ (G) mRNA level in peri-infarct area of control and Piezo1 CKO mice within the first 72 h after MCAO/R. H Flow cytometry analysis of TNF-α positive cells in MACS microglia isolated from peri-infarct area of control and Piezo1 CKO mice 72 h after MCAO/R. I Quantitative analysis of TNF-α positive cells in panel H. H Flow cytometry analysis of IFN-γ positive cells in MACS microglia isolated from peri-infarct area of control and Piezo1 CKO mice 72 h after MCAO/R. I Quantitative analysis of IFN-γ positive cells in panel H. L Volcano plot analysis showing dysregulated genes related to IFN-γ signaling performed in MACS microglia isolated from control and Piezo1 CKO mice at 72 h after MCAO/R. M Pathway enrichment analysis using Reactome datasets displaying the top 20 terms compared between control and Piezo1 CKO mice at 72 h after MCAO/R. N Flow cytometry analysis of CXCL10 positive astrocytes in astrocytes and control of Piezo1 CKO microglia co-culture system treated by TNF-α and/or IFN-γ antibodies upon OGD/R condition. O Quantitative analysis of CXCL10 positive cells in panel N. Data are expressed as mean ± SD. Two-tailed unpaired t-test for A, C, E, I and K. Two-way ANOVA with Sidak’s post hoc test in F, G. One-way ANOVA followed by Dunnett’s post hoc test for O. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: no significant.

Article Snippet: Paired-end sequencing libraries were performed using a mRNA NEBNext Poly selection Library Prep Kit (Illumina, Los Angeles, CA, USA). cDNA sequence was then conducted using the Illumina NovaSeq 6000 sequencing platform with multiplex paired-end 150 base pairs reads.

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Isolation, Co-Culture Assay, Two Tailed Test