GSM2498239: CFPAC-1 negative rep1; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy® Mini Kit (Qiagen, 74104), and purified with DNase I (Qiagen, 79254). RNA was then quantified using the NanoDrop2000 spectrophotometer (Thermo Scientific). 1μg quantified total RNA was taken and enriched for PolyA using Oligo(dT) Microparticles. Transcriptome library for sequencing was constructed according to SureSelect Strand-Specific RNA Library Prep Kit protocol outlined in “SureSelect Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing” (Cat #5500-0135). Briefly, the mRNA was fragmented for 2 minutes at elevated temperature (94oC) in the presence of divalent cations and first strand cDNA was synthesized. The single stranded cDNA was cleaned up using HighPrep (Magbio, Cat #AC-60050). Strand Specificity is maintained by the addition of Actinomycin D at this step. Second strand cDNA was synthesized and end repaired using Second Strand Synthesis + End Repair mix. The cDNA was cleaned up using HighPrep (Magbio, Cat #AC-60050). Adapters were ligated to the cDNA molecules after addition of “A”- base. HighPrep cleanup was performed post ligation. The library was indexed and enriched for adapter ligated fragments using 10 cycles of PCR. The prepared library was quantified using Qubit and validated for quality by running an aliquot on D1000 Tapes. Finally, the adapter positive fragments were quantified using KAPA qPCR quantification kit (KAPA Biosystems; Cat #KK4824).