title: Illumina HiSeq 4000 paired end sequencing; Single cell RNA sequencing of paired non small cell lung carcinoma tissue and peripheral blood from 5 donors
design: Single cell RNA sequencing of paired non small cell lung carcinoma tissue and peripheral blood from 5 donors
library_construction_protocol: 1. Cut tumour into small pieces and dissociate the tumour tissue using the Human Tumor Dissociation Kit (Milteny Biotec). 2. Collect whole blood in an EDTA K3-tube. Separate PBMC using PANCOLL lymphocyte separation medium (Pan Biotech).  3. Freeze single cell suspension in Gibco Recovery Cell Culture Freezing Medium (Thermo Fisher Scientific) for shipping and long term storage. 4. Immediately prior to cell processing thaw cells by adding complete media dropwise to frozen vial and resuspend in RPMI 1640 + 10% FBS with the addition of 15 U/ml of RNase-free DNase I.  5. Count cells using viability dye.  6.  Before loading stain each sample individually with Sample Tag.  1. Load cells in BD Rhapsody cartridge (~30,000 cells / cartridge) by gravity flow. 2. Wash BD Rhapsody Cell Capture Beads with sample buffer. 3. Load BD Rhapsody Cell Capture Beads in cartridge. 4. Wash cartridge twice with sample buffer. 5. Lyse cells with lysis buffer containing fresh DTT.  6. Bind released RNA to beads by incubating.  7. Retrieve beads from cartridge by applying magnet. 8. Collect beads by removing magnet and washing into collection tube.  9. Wash collected beads twice. 10. Perform bulk reverse transcription on beads in a heat block at 37 °C for 30 minutes.  11. Treat sample with Exonuclease I at 37 °C for 30 minutes.  12. Inactivate enzymes by heating to 80 °C for 30 minutes  1. Perform targeted amplification using the BD Rhapsody Immune Response Panel Hs and the Sample Tag primer mix. 2. Purify PCR1 products with Agencourt AMPure XP magnetic beads.  3. Perform PCR2 amplification on Targeted PCR1 products and Sample Tag PCR1 products separately.  4. Purify PCR2 products separately with Agencourt AMPure XP magnetic beads.  5. Quantify amplified libraries using Qubit dsDNA Quantification Kit. 6. Perform Illumina adapter addition and final amplification on Targeted PCR2 products and Sample Tag PCR2 products separately.  7. Purify final libraries separately with Agencourt AMPure XP magnetic beads.  8. Quantify amplified libraries using Qubit dsDNA Quantification Kit. 9. QC libraries by running Bioanalyzer High Sensitivity DNA Analysis (Agilent).  10. Pool Targeted library and Sample Tag library for sequencing.