GSM3265040: TAM-NSCLC TAM-99-S; Homo sapiens; RNA-Seq
Resected tissue was dissagregated mechanically and enzymatically (liberase DL and DNAseI, 15 min 37C). FACS sorted macrophages (DAPIneg, CD45pos, CD14pos, HLA-DRpos) were stored at -80C in trizol. RNA was extracted using miRNeasy micro kit (QIAGEN) following manufacturer instructions and amplified using SEQR SeqPlex RNA Amplification kit (Sigma) Amplified dsDNA was subjected to library preparation using TruSeq Nano DNA library prep kit (Illumina). Briefly: Samples were subjected to End-Repair during 30 min at 30C. Size selection was performed using Agencourt AMPure XP beads in two steps: Removal of large DNA fragments (>450 bp) followed by removal of small DNA fragments (<150 bp). 3' ends of 150-450bp DNA fragments were adenylated using A-Tailing Mix during 30 min at 37C. Then, Illumina adapters were ligated using Ligation Mix 2, Resuspension Buffer and the appropriate barcoded adapter in each case. As a last step, two sets of clean ups were performed using AMPure XP Beads. DNA was eluted and PCR amplified using PCR Primer Cocktail and Enhanced PCR Mix as follows: 3 min at 95C, and 8 cycles of 20 sec at 98C, 15 sec at 60C and 30 sec at 70C. As a final step, the final amplified libraries were cleaned up using Sample Purification Beads. The size of the library was determined by HS-DNA Agilent Bioanalyzer (150-430 bp). The amount of library was determined by Nanodrop and dsDNA Quantifluor.