biostudies-arrayexpressJinhua ZhangMus musculusIllumina NovaSeq 6000https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16060RNA-sequencing analyses were performed in CD8+ T cells sorted from MC38 tumors of WT and Lypd6b−/− mice， To investigate the effects of Lypd6b gene knockout on CD8+ T cells in the colon cancer tumor microenvironment. Abstract Lypd6b is a newly discovered molecule associated with neuromodulation. However, the effects of Lypd6b on the tumor microenvironment and its impact on CD8+ T cell-mediated antitumor immunity remain unknown. Here, we observe that Lypd6b expression is increased significantly in colorectal cancer (CRC) tumor tissues compared to normal tissues. Lypd6b is mainly expressed in CD8+ T cells in tumor tissues. Lypd6b knockout (Lypd6b−/−) mice are resistant to AOM/DSS-induced tumorigenesis. Furthermore, global deficiency or CD8+ cell deficiency of Lypd6b (CD8Lypd6b−/−) inhibits MC38 or CMT-93 tumor growth and promotes the infiltration of CD8+ T cells. Mechanistically, Lypd6b deficiency promotes activation and function of CD8+ T cells in anti-tumor response with increased glycolysis and reduced oxidative phosphorylation (OXPHOS) in a PI3K/mTOR/LDHA pathway-dependent manner. Notably, Lypd6b deficient CD8+ T cells have a more potent antitumor effect when combined with anti-PD1 antibody. Thus, Lypd6b as a negative regulator for T cell immunity promotes CRC development, providing a molecular target with therapeutic potential in CRC.biostudies-arrayexpressNucleic Acid Extraction - Add 350 μL Buffer RLT (Add 10μL β-ME into 1mL Buffer RLT before use), mix by vortexing or pipetting to dissolve the cell, stay at room temperature for 5 min. 2.1.3 Lysis by TRIzol: Add 1/5 volume of Chloroform/iso-amyl alcohol（24:1）to the defrosted sample, centrifuge at 12,000xg for 10min and transfer the supernatant to the new centrifuge tube. 2.2 Add the same volume of 75% ethanol and mix by pipetting. 2.3 Add the sample to the RNeasy MinElute spin column, centrifuge at 10,000×g for 15 s. Discard the filtrate. 2.4 Add 350 μL RW1, centrifuge at 10,000×g for 15 s, discard the filtrate. 2.5 Add 10 µL DNase I and 70 µL Buffer RDD, stay at room temperature for 15 min. 2.6 Add 350 μL RW1, centrifuge at 10,000×g for 15 s. Discard the filtrate. 2.7 Add 500 μL RPE, centrifuge at 10,000×g for 15 s. Discard the filtrate. 2.8 Add 500 μL RPE, centrifuge at 10,000×g for 15 s. Discard the filtrate. Transfer the RNeasy MinElute spin column to new collection tube, centrifuge at full speed for 5 min. 2.10 Transfer RNeasy MinElute spin column to a new 1.5 mL centrifuge tube, add 20 μL RNase-free water, centrifuge at full speed to elute RNA. 2.11 Check RNA quality as required.Growth Protocol - Six- to eight-week-old female C57BL/6 and Lypd6b-/- mice were selected, and 7×105 MC38 cells. mice were maintained in pathogen-free and humidity- and temperature-controlled (22 ± 2 °C) conditions with a 12-h light/dark cycle (lights on from 7:00 to 19:00), and 50–60% relative humidity at the National Institute of Biological Science.Naïve CD8+ T lymphocytes were isolated from mouse tumors by negative selection using the Naïve CD8+ T Cell Isolation Kit (BioLegend) according to the manufacturer’s instructions.Sequencing - 1. Introduction RNA-Seq (Quantification) uses a new generation of high-throughput sequencing technology to study the gene expression pattern of eukaryotic cells and provide accurate digital expression profile detection by sequencing and comparing transcripts. Transcriptome research is the basis and starting point of gene function and structure research. Through the new generation of high-throughput sequencing, almost all transcripts of a specific tissue or organ of a species can be obtained comprehensively and quickly. 2.Experimental Procedure 2.1 Sample QC Select the corresponding testing methods for quality inspection according to the requirements of samples and products. 2.2 mRNA Isolation A certain amount of RNA samples are denatured at suitable temperature to open their secondary structure, and mRNA is enriched by oligo (dT) -attached magnetic beads. 2.3 mRNA Fragmentation The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented. 2.4 cDNA Synthesis Prepare the first-strand synthesis reaction system, and set up the reaction program, synthesize the first- strand cDNA, prepare the second-strand synthesis reaction system, and set up the reaction program to synthesize the second-strand cDNA. 2.5 End Repair, Add A and Adaptor Ligation After the reaction system and program are configured and set up, double-stranded cDNA fragments are subjected to end-repair, and then a single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments. The reaction system and program for adaptor ligation are subsequently configured and set up to ligate adaptors with the cDNAs. 2.6 PCR The PCR reaction system and program are configured and set up to amplify the product. 2.7 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 2.8 Circularization Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. 2.9 Sequencing Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).Sample Collection - Naïve CD8+ T lymphocytes were isolated from mouse spleens by negative selection using the Naïve CD8+ T Cell Isolation Kit (BioLegend) according to the manufacturer’s instructions.Library Construction - 2.1 Sample QC The corresponding sample quality control protocol will be selected depending upon sample types and product requirements. 2.2 RNA Denaturation A certain amount of RNA samples. Add Oligo-dT Primer into the sample and then put the sample on PCR instrument for RNA denaturation. 2.3 cDNA Synthesis The reaction system is configured for reverse transcription. First-strand cDNA is synthesized with SMART-SeqII. The reaction system is configured for PCR amplification to process preamplification of cDNAs. 2.4 Library Construction Qualified amplified cDNAs use transposon-based method for further library construction. 2.5 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 2.6 Circularization Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. 2.7 Sequencing Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).Sequencing - RNA-sequencing analyses were performed in CD8+ T cells sorted from MC38 tumors of WT and Lypd6−/− mice. Total RNA was extracted with RNeasy Mini Kit (QIAGEN, Dusseldorf, Germany).Sample Collection - Naïve CD8+ T lymphocytes were isolated from mouse tumor tissues by negative selection using the Naïve CD8+ T Cell Isolation Kit (BioLegend, USA) according to the manufacturer’s instructions.Library Construction - .2 mRNA Isolation Take the desired amount of RNA samples. mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. 2.3 Globin mRNA Removal The reaction system is configured. Use Globin clear kit to remove Globin mRNA. 2.4 mRNA Fragmentation The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented. 2.5 cDNA Synthesis Add the preprepared first-strand synthesis reaction mixture into the fragmented RNAs. The reaction program is set up for synthesizing first-strand cDNAs. The reaction system and program are configured and set up for second-strand cDNA synthesis, dUTP instead of deoxythymidine triphosphate (dTTP) is used. 2.6 End Repair, Add A and Adaptor Ligation After the reaction system and program are configured and set up, double-stranded cDNA fragments are subjected to end-repair, and then a single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments. The reaction system and program for adaptor ligation are subsequently configured and set up to ligate adaptors with the cDNAs. 2.7 PCR The PCR reaction system and program are configured and set up to amplify the product. 2.8 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 2.9 Circularization Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. 2.10 Sequencing Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).Sample Treatment - CD8+ T cells from tumors of WT and Lypd6b−/− mice were sorted and activated for 48 h in the presence of CD3 and CD28 antibodies.Nucleic Acid Extraction - Add 350 μL Buffer RLT (Add 10μL β-ME into 1mL Buffer RLT before use), mix by vortexing or pipetting to dissolve the cell, stay at room temperature for 5 min.Lysis by TRIzol: Add 1/5 volume of Chloroform/iso-amyl alcohol（24:1）to the defrosted sample, centrifuge at 12,000xg for 10min and transfer the supernatant to the new centrifuge tube. 2.2 Add the same volume of 75% ethanol and mix by pipetting. 2.3 Add the sample to the RNeasy MinElute spin column, centrifuge at 10,000×g for 15 s. Discard the filtrate. 2.4 Add 350 μL RW1, centrifuge at 10,000×g for 15 s, discard the filtrate. 2.5 Add 10 µL DNase I and 70 µL Buffer RDD, stay at room temperature for 15 min. 2.6 Add 350 μL RW1, centrifuge at 10,000×g for 15 s. Discard the filtrate. 2.7 Add 500 μL RPE, centrifuge at 10,000×g for 15 s. Discard the filtrate. 2.8 Add 500 μL RPE, centrifuge at 10,000×g for 15 s. Discard the filtrate. 2.9 Transfer the RNeasy MinElute spin column to new collection tube, centrifuge at full speed for 5 min. 2.10 Transfer RNeasy MinElute spin column to a new 1.5 mL centrifuge tube, add 20 μL RNase-free water, centrifuge at full speed to elute RNA. 2.11 Check RNA quality as required.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesSequence Alignment - Data filtering The sequencing data was filtered with SOAPnuke [1] by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system (https://biosys.bgi.com). RNA identification The clean reads were mapped to the reference genome using HISAT2 [2]. Bowtie2[3] was applied to align the clean reads to the gene set, in which known and novel, coding and noncoding transcripts were included. Gene Quantification Differential Expression Analysis Expression level of gene was calculated by RSEM (v1.3.1) [4]. The heatmap was drawn by pheatmap (v1.0.12) [5] according to the gene expression difference in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.34.0) [6] (or DEGseq（v1.48.0）[7] or PoissonDis[8])with Q value ≤ 0.05 (or FDR ≤ 0.001). Gene Annotation To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expression gene was performed by Phyper based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05)Data Transformation - The sequencing data was filtered with SOAPnuke [1] by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system (https://biosys.bgi.com).UnknownTranscriptomicsGenomicsProteomicsDNBSEQ-T7The solution conformation of the self-complementary RNA-DNA hybrid hexamer 5'-[r(GCA)d(TGC)]2 is investigated by NMR spectroscopy and restrained molecular dynamics. The 1H-NMR spectrum is assigned in a sequential manner using two-dimensional homonuclear Hartmann-Hahn and nuclear Overhauser enhancement spectroscopy. From the latter a set of 178 approximate interproton distance restraints are determined and used as the basis of a structure refinement by restrained molecular dynamics. Eight independent calculations are carried out, four from a classical A-type geometry and four from a classical B-type one. Convergence is achieved to very similar A-type structures with an average atomic root mean square difference between them of 1.0 +/- 0.2 A. The converged structures exhibit variations in helical parameters similar to those found previously for the analogue RNA hexamer 5'-r(GCAUGC)2 [(1988) Biochemistry 27, 1735-1743].RNA-seq of coding RNAMus musculusLypd6b depletion promotes CD8+ T cell-mediated anti-tumor immunity via metabolic reprogramming in CRCJinhua ZhangfalseRNA-seg of CD8+ T cells from colorectal cancer tumor tissues in wildtype and Lypd6−/− miceRNA-sequencing analyses were performed in CD8+ T cells sorted from MC38 tumors of WT and Lypd6b−/− mice， To investigate the effects of Lypd6b gene knockout on CD8+ T cells in the colon cancer tumor microenvironment. Abstract Lypd6b is a newly discovered molecule associated with neuromodulation. However, the effects of Lypd6b on the tumor microenvironment and its impact on CD8+ T cell-mediated antitumor immunity remain unknown. Here, we observe that Lypd6b expression is increased significantly in colorectal cancer (CRC) tumor tissues compared to normal tissues. Lypd6b is mainly expressed in CD8+ T cells in tumor tissues. Lypd6b knockout (Lypd6b−/−) mice are resistant to AOM/DSS-induced tumorigenesis. Furthermore, global deficiency or CD8+ cell deficiency of Lypd6b (CD8Lypd6b−/−) inhibits MC38 or CMT-93 tumor growth and promotes the infiltration of CD8+ T cells. Mechanistically, Lypd6b deficiency promotes activation and function of CD8+ T cells in anti-tumor response with increased glycolysis and reduced oxidative phosphorylation (OXPHOS) in a PI3K/mTOR/LDHA pathway-dependent manner. Notably, Lypd6b deficient CD8+ T cells have a more potent antitumor effect when combined with anti-PD1 antibody. Thus, Lypd6b as a negative regulator for T cell immunity promotes CRC development, providing a molecular target with therapeutic potential in CRC.2025-12-11T00:00:00Z2025-12-11T02:01:57.205Z2025-11-13T14:43:39.621ZE-MTAB-160602456957ERP184097EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.21203/rs.3.rs-5050970/v1