Smarter ultra low input rna for sequencing hv kit

RNA-seq was performed in three independent biological replicates for nuc^+XopD and nuc^−XopD. For each sample, approximately 500 pg nuclear RNA was extracted from ∼10⁵ nuclei using the RNeasy Plus Micro Kit (Qiagen) and further treated with DNase I (0.05 U per μl, Thermo Fisher Scientific) for 30 min at 37 °C to remove any contaminating genomic DNA. The double-stranded (ds) cDNA was synthesized from ∼500 pg RNA by two rounds of linear amplification using the SMARTer Ultra Low Input RNA for Illumina Sequencing-HV kit (Clontech) according to the manufacturer’s instructions. The concentration and yield of the amplified cDNA was determined using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen). RNA-seq libraries were prepared using the Low Input Library Prep Kit (Clontech) according to the manufacturer’s instructions. The quality and quantity of the RNA-seq libraries was examined using the High Sensitivity DNA Kit (Agilent). Sequencing was performed on NovaSeq 6000 system (Novogene, UK). From 33.2 to 40.9 millions of 2× 150-bp paired-end reads that passed the Illumina quality control filter were collected for each sample (Supplementary Table 8).

In total, 1110–4566 GFP-expressing viable cells were sorted from E9.5 embryos carrying the Mef2c-AHF-GFP allele^{33 (link)} by FACS using a Beckman Coulter MoFlo AstriosEQ with Summit 6.2.7.16492 software. GFP was detected with the 488 nm laser and a 513/26 band pass filter, and DAPI as a viability dye was detected with the 405 nm laser and a 448/59 band pass filter. Cells were collected into Eppendorf DNA LoBind tubes containing lysis buffer, and RNA isolated using a Qiagen RNeasy Mini kit. RNA quality was assessed using RNA pico chips on an Agilent 2100 Bioanalyzer. Library preparation (SMARTer Ultra Low Input RNA for Illumina Sequencing - HV kit) and sequencing (Illumina HiSeq4000) were performed by the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics. The samples were processed sequenced in two batches. Batch 1 contained three control and five hypoxia samples, and batch 2 contained two repeated control samples with five ID samples.