RNA-seg of CD8+ T cells from colorectal cancer tumor tissues in wildtype and  Lypd6−/− mice
RNA-seg of CD8+ T cells from colorectal cancer tumor tissues in wildtype and  Lypd6−/− mice
 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.  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. Six- to eight-week-old female C57BL/6 mice were selected, and 7×105 MC38 cells.After the tumor had grown to a certain size, dissected and mechanically sheared the tumor tissues, then they were digested in DMEM medium with DNase I (50 μg/mL) and Collagenase IV (1 mg/mL) (Millipore-Sigma) for 60 min at 37°℃ accompanied by gentle shaking. The cells were collected after filtering cell suspension through a 40 μm cell strainer twice. 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.  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.  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). 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. 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. .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). 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).  