Truseq sr cluster kit v3

RNA sequencing analysis was performed in 24 samples. The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories) was used to generate first-strand cDNA from 750 pg total-RNA. Double-stranded cDNA was amplified by LD PCR (12 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina). 150 pg of input cDNA was tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems). Equimolar amounts of each library were pooled, and the pools then used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide. Image analysis and base calling resulted in bcl files, which were converted into fastq files with the bcl2fastq v2.18 software. The sequencing data are available in the Gene Expression Omnibus database under accession number GSE179568.

Total RNA (1.0 μg each) from each sample was used to construct miRNA library with an unique index using the TruSeq Small RNA Sample Preparation kit (Illumina, San Diego, CA) according to the manufacturer's instruction. PCR amplification was performed for 11 cycles and libraries with unique indices were purified individually using gel purification. Quantitative real time PCR (qPCR) was performed for library quantification using the StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA) and KAPA SYBR Fast ABI Prism qPCR kit (Kapa Biosystems, Woburn, MA). The diluted libraries were loaded on cBot (Illumina) for cluster generation using the TruSeq SR Cluster kit v3 (Illumina). Sequencing was performed on the HiScan SQ system (Illumina) using the TruSeq SBS kit v3 (50 cycles, Illumina). Real-time analysis and base calling was performed using the HiSeq Control Software Version 1.4.8 (Illumina).

Construction of libraries was done using the NEB-Next multiplex Small RNA Preparation Kit (New England BioLabs, USA) according to the manufacturer’s instructions using 150 ng RNA per sample. Next, individual libraries were adjusted to 2 nM and pooled before denaturation and dilution according to Illumina’s instructions. cDNA libraries were prepared from gel-extracted size-selected RNA with a median length of 140 nucleotides. Library quality for miRs was determined by TapeStation and BioAnalyzer. APhiX control library was spiked in, to determine the sequencing error rate (<1%). The diluted libraries (20 pM) were loaded onto a 4-lane flow cell for cluster formation using the TruSeq™ SR Cluster Kit v3 (Illumina). Sequencing was performed on an Illumina NextSeq 500 system. Libraries were sequenced from the 5′- end using a read length of 30 nt and results converted into fastq files with Casava 1.8.2 (Illumina) using BaseSpace software and downloaded as Fastq files for alignment and further analysis.

Cells were cross-linked with 1% methanol-free formaldehyde for 10 min. After quenching with glycine, cells were washed three times with PBS. The cell pellet was treated with 40 U MNase for 5 min at 37°C and then stopped with 10× Covaris buffer (Covaris), and chromatin was sheared for 15 min with the Covaris S2 device (burst 200; cycle 20%; intensity 8). Immunoprecipitation was performed for approximately 5 × 10⁶ cells with anti-H3 antibody (Abcam ab1791, lot GR103864-1). Then chromatin was treated with RNase A and Proteinase K. Purified DNA was cloned into Illumina libraries with the NEBNext ultra library preparation kit (NEB). Paired-end reads were sequenced using Illumina HiSeq 2000.
RNA-seq was performed using total RNA extracted using a DNA-Free RNA kit (Zymo Research) as detailed in the Supplemental Methods. Libraries were prepared from RNA of WT and DKO ESCs using the TruSeq RNA sample preparation kit v2 (Illumina), clustered on cBot (Illumina) using TruSeq SR Cluster Kit v3 and sequenced by single-read 50-bp mode on a HiSeq 2000 v3 platform according to Illumina's instructions. RNA-seq analysis was performed in Genomatix (Genomatix GmbH) as detailed in the Supplemental Methods.

Total RNA input of 100 ng was used for library construction with the TruSeq Stranded mRNA LT kit—Set A (Illumina, RS-122-2101), together with the SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014), according to the manufacturer’s protocol. The quality of mRNA libraries was assessed for adapter and heterodimer presence using the DNA 1000 microfluidic chip (Agilent, 5067-1504), while library molarity was measured using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P11496). All 32 sequencing libraries were then normalized, pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered using the TruSeq SR Cluster Kit v3 (Illumina, GD-401-3001), and sequenced with TruSeq SBS Kit v3 reagents (Illumina, FC-401-3002) on a HiSeq 2000 Sequencing System (Illumina) with single index, single-read reads at 1 × 51 bp length (Read parameters: Rd1: 52, Rd2: 7), reaching an average depth of 24.9 million Pass-Filter reads per sample (13.1% CV).