title: Illumina HiSeq 4000 sequencing; RNA-Seq Analysis of Human Lung Cancer Cell Line A549 Following GSE1 Knockdown: Differential Gene Expression and Pathway Analysis Against siRNA Controls
design: RNA-Seq Analysis of Human Lung Cancer Cell Line A549 Following GSE1 Knockdown: Differential Gene Expression and Pathway Analysis Against siRNA Controls
library_construction_protocol: Sample Collection Protocol for RNA Sequencing in A549 Cells 1. Cell Plating: A549 human lung adenocarcinoma cells are plated at a density of 2 × 10^5 cells per well in 6-well plates. Cells are cultured in DMEM medium supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin. 2. Cell Culture: Cells are incubated for 24 hours at 37°C in a humidified atmosphere containing 5% CO2 to allow for cell attachment and growth. 3. Transfection: Post 24-hour incubation, cells are transfected with either GSE1-targeting siRNA or negative control siRNA using Lipofectamine 2000, following the manufacturer's guidelines. 4. Post-Transfection Incubation: Cells are again incubated for 48 hours under the same culture conditions to allow for gene knockdown. 5. Validation of Knockdown: The efficiency of GSE1 gene knockdown is confirmed via western blotting prior to RNA extraction. 6. RNA Isolation: Post-validation, cells are washed twice with phosphate-buffered saline (PBS) and subjected to total RNA extraction using TRIzol reagent as per the manufacturer's instructions. 7. RNA Quantification: The extracted RNA is then quantified and assessed for purity using a NanoDrop One Microvolume UV-Vis Spectrophotometer. Nucleic Acid (RNA) Extraction Protocol from A549 Cells 1. Cell Washing: After 48 hours of post-transfection incubation, A549 cells in 6-well plates are washed twice with cold phosphate-buffered saline (PBS) to remove any residual media and non-adherent cells. 2. Reagent Preparation: TRIzol reagent (Invitrogen) is brought to room temperature before use. 3. RNA Isolation: Approximately 1 mL of TRIzol reagent is added directly to each well, and cells are lysed by pipetting up and down. The lysate is collected and transferred to a microcentrifuge tube. 4. Phase Separation: The cell lysate is incubated at room temperature for 5 minutes to permit the complete dissociation of nucleoprotein complexes. Then, it is centrifuged with chloroform for phase separation.  5. RNA Precipitation: The aqueous phase containing RNA is transferred to a new tube. RNA is precipitated by adding isopropanol, followed by a brief incubation and centrifugation. 6. RNA Washing: The RNA pellet is washed with 75% ethanol to remove impurities, followed by a centrifugation step. 7. RNA Resuspension: The RNA pellet is air-dried and then resuspended in a suitable volume of RNase-free water. 8. RNA Quantification: The quantity and quality of the extracted RNA are assessed using a NanoDrop One Microvolume UV-Vis Spectrophotometer to ensure adequate yield and purity for downstream applications.   RNA Library Construction Protocol for RNA-Seq 1. Quality Control: Prior to library construction, ensure that the isolated RNA has high purity and integrity, usually assessed via NanoDrop and possibly an RNA Integrity Number (RIN). 2. RNA Quantification: Measure 500 ng of high-quality total RNA as the starting material for the library preparation. 3. Kit Preparation: Use the TruSeq RNA Sample Preparation Kit (Illumina) and prepare the reagents as per the manufacturer's guidelines. 4. RNA Fragmentation: The RNA is first fragmented into smaller pieces, either enzymatically or thermally, depending on the kit's protocol. 5. First-Strand cDNA Synthesis: Use reverse transcriptase to synthesize the first strand of cDNA from the fragmented RNA, using random primers. 6. Second-Strand cDNA Synthesis: A second cDNA strand is synthesized to form a stable double-stranded cDNA fragment. 7. End Repair: The double-stranded cDNA fragments undergo end-repair to create blunt ends, facilitating adapter ligation. 8. Adapter Ligation: Adapters containing the sequences necessary for downstream sequencing are ligated to the repaired cDNA ends. 9. PCR Amplification: The adapter-ligated cDNA is amplified using PCR to produce sufficient material for sequencing. This step also incorporates index sequences that allow for multiplexing. 10. Library Validation: Confirm the quality and size distribution of the prepared library, often using a Bioanalyzer or TapeStation. 11. Library Quantification: Quantify the final library concentration to ensure it meets the requirements for sequencing, typically using qPCR or fluorometric methods like Qubit. 12. Sequencing: Load the validated and quantified library into the Illumina HiSeq 4000 sequencing instrument for downstream high-throughput sequencing.