title: GSM8141540: DU145_shSOX4_2; Homo sapiens; RNA-Seq
library_name: GSM8141540
library_construction_protocol: For RNA-seq, 1 μg of total RNA was subjected to library synthesis using the TruSeq V2 RNA-Seq kit. 1 μg of total RNA was enriched for the Poly-A mRNA and reverse transcribed to double-stranded cDNA. The cDNA was end repaired, adenylated, and ligated with Illumina indexes prior to 12 cycles of PCR. Sequencing was performed using 12pM hybridization to a 2x100 paired end flow cell. For the ATAC-seq assay, 50000 cells were centrifuged at 500 g for 5 min at 4℃, and the supernatant was removed. Cells were washed once with cold PBS. Subsequently, the cells were again centrifuged at 500 g for 5 min at 4℃ and the supernatant was removed. The cells were then suspended in cold lysis buffer. Next, the cells were again centrifuged at 500 g for 10 min at 4℃ and the supernatant was removed. The transposition reaction system was performed using Tn5 transposase. The cell nuclear content was added to the transposing reaction system mixture, and the DNA were purified after incubation at 37℃ for 30 min. The PCR system was performed with purified DNA, and PCR amplification was performed. For ATAC-seq, 40 000 nuclei were pelleted and resuspended with transposase, for 30 minutes at 37 ℃. The transposed DNA fragments were purified immediately after with a MinElute PCR Purification Kit (Qiagen). After Samples were PCR-amplified using 1X NEBNext High-Fidelity PCR Master Mix (New England Biolabs, MA). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd.) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligoattached magnetic beads.First strand cDNA was synthesized and second strand cDNA synthesis was subsequently performed. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. ﻿ATAC was performed according to ATAC-seq protocol by Biomarker Technologies Biotechnology Ltd. In brief, native nuclei were purified from cell as previously described (Buenrostro et al.,2015). The Nextera DNA Library Preparation Kit (Illumina) was used to perform the transposition according to the manufacturer's manual. 40 000 nuclei were pelleted and resuspended with transposase, for 30 minutes at 37 ℃. The transposed DNA fragments were purified immediately after with a MinElute PCR Purification Kit (Qiagen). After Samples were PCR-amplified using 1X NEBNext High-Fidelity PCR Master Mix (New England Biolabs, MA). Subsequent libraries were purified with the MinElute PCR Purification Kit (Qiagen) and subjected to sequencing on Illumina Novaseq 6000 using PE150.