KAPA Stranded mRNA-Seq Kit

Protocol tips

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	This protocol has been validated for library construction from 100 ng – 4 μg intact, total RNA, in ≤50 µL of RNase-free water.

Protocol tips
This protocol has been validated for library construction from 100 ng – 4 μg intact, total RNA, in ≤50 µL of RNase-free water.

Publication protocol

Total RNA was purified using the RNeasy Plus Mini kit (Qiagen). For qRT-PCR (Figure 5D and S5E), RNA was reverse-transcribed to cDNA using High Capacity cDNA RT kit (Applied Biosystems), and amplified in TaqMan assays on a 7500 Fast Real-time PCR System (Applied Biosystems). mRNA-Seq libraries were constructed using 4 μg total RNA with the Stranded mRNA-Seq Kit (KAPA Biosystems). 12 samples were run on a single flow cell: SKBR-3 and BT474 parental lines each treated with DMSO, 300nM lapatinib, 300nM JQ1, and the combination for 48 h, and lapatinib-resistant SKBR-3 treated for 8 days with 300nM lapatinib, lapatinib+300nM JQ1, or lapatinib+1µM I-BET151, in addition to parental SKBR-3 cells. 75-cycle single-end sequencing runs were generated with an Illumina NextSeq-500. QC-passed reads were aligned to the human reference genome (hg19) using MapSplice. The alignment profile was determined by Picard Tools v1.64. Aligned reads were sorted and indexed using SAMtools and translated to transcriptome coordinates and filtered for indels, large inserts, and zero mapping quality using UBU v1.0. Transcript abundance estimates for each sample were performed using an Expectation-Maximization algorithm.

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Manufacturer protocol

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