title: GSM2936446: Tumor_Lungs_FIGF_FIGF_5-3; Homo sapiens; RNA-Seq
library_construction_protocol: The mRNA selection and whole transcriptome amplification were performed as previously described with some modifications. A primer 5'-phosphorylated oligo-dT24 (pdT24) was used to select mRNA from total RNA. For denaturation and primer annealing, total RNA was mixed with dNTPs (25 mM) and pdT24, and put in a preheated 68°C PCR machine for 5 min and immediately placed on ice. Reverse transcription was performed with the addition of the First-Strand Buffer, DTT, RNase Inhibitor, and 10 U/μl Superscript Reverse Transcriptase III. The double-strand cDNA was generated by the addition of 30 μl mix containing 10x second100 strand buffer, dNTPs (25 mM), E.coli DNA ligase (10 U/μl), RNaseH, and DNA Polymerase (10U/μl), followed by 3 hr incubation at 16°C and 5 min at 70°C. The product was purified with the Genomic DNA Clean and Concentrator kit (Zymo). Then, DNA end-bluting, 5'-end phosphorylation, and ligation were performed with End-It DNA End-Repair Kit (Epicentre) and T4 DNA ligase (Epicentre). The product was directly amplified by adding the mixture of the Reaction Buffer and DNA polymerase from REPLI-g UltraFast Kit (Qiagen) in 15:1 ratio. The amplification reaction was carried at 30°C for 2 hr. The amplified product was purified using the same Genomic DNA Clean and Concentrator kit (Zymo). When eluted in 50 μl elution buffer, typically 5-10 μg of mRNA-derived cDNA amplicons were obtained. The amplicons were then evaluated for quantity, coverage, and purity by PCR using a set of primers for housekeeping genes (GAPDH, B2M), cDNA 5' end (CREB5), and gDNA (10p). cDNA libraries were constructed using Nextera XT DNA Sample preparation kit (Illumina) according to the manufacturer's recommendations with minor modifications. Briefly, cDNA amplicons were fragmented and barcoded by tagmentation using Tn5 DNA transposase. Tagmented cDNA were amplified with a limited-cycle PCR program. During the PCR step, indices 1 and 2 were added to allow for cluster generation and multiplexed sequencing. The extension time was increased from 30s to 60s. The amplified cDNA libraries were purified and cleaned using 0.6x Agencourt AMPure XP (Beckman Coulter) and fresh ethanol, and eluted in TE buffer. The quality of sequencing libraries was assessed using a high-sensitivity DNA chip (Agilent). Finally, the libraries were sequenced deeply using the HiSeq2000 sequencer (Illumina). For the single cell RNA-Seq data, the cells were processed according to the 10xGenomics sample preparation protocol (Chromium Single Cell 3' v2 Reagent Kit, 10xGenomics).
attributes.tag: GEO Accession
attributes.value: GSM2936446