Smarter ultra low input rna kit for sequencing

Total RNA isolation was performed using an RNeasy plus Micro Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using a SMARTer Ultra Low Input RNA Kit for Sequencing (Clontech). cDNA libraries were generated with 6x10³ LSK cells and GMPs using a NEBNext Ultra DNA Library Prep Kit (New England BioLabs) according to the manufacturer’s indications. The RNA-sequence reads were aligned using TopHat 1 (version 2.0.13; with default parameters) and levels of gene expression were quantified using Cufflinks (version 2.2.1).

Due to low RNA yields, the SMARTer Ultra Low Input RNA kit for sequencing (v. 3; Clontech Laboratories, Inc., Mountain View, CA) was used for cDNA synthesis with a template input amount of 5 ng. The Illumina TruSeq Stranded mRNA-Seq HT sample preparation kit (Illumina, San Diego, CA) and its workflow were used for preparation of dual-indexed transcriptome libraries. A normalized input amount of 20 ng total RNA was used for the library prep. Final library products were measured by quantitative PCR using the Kapa Illumina Library quantification kit (Kapa Biosystems, Boston, MA), pooled at equal molar amounts, and sequenced on two lanes of a HiSeq 2500 8-lane flow cell (Illumina) to produce paired 100-bp reads. Trimmomatic v. 0.32 (69 (link)) was used to remove any remaining Illumina adaptor sequence and low-quality bases from the reads, and FastQC v. 0.11.2 (70 ) was used to assess the quality of reads before and after trimming.

Total RNAs were extracted from pooled HSCs and fibroblasts of 3–5 mice using the PureLink RNA Micro Kit (three samples per each group). Total RNAs were amplified using a SMARTer Ultra Low Input RNA Kit for Sequencing (Clontech, Mountain View, CA, USA). RNA-seq libraries were prepared according to the manufacturer’s protocol (Illumina, San Diego, CA, USA). Libraries were single-endsequenced on a GAIIx sequencer (Illumina, San Diego, CA, USA). Reads were aligned to the mm10 mouse genome using STAR. Aligned read files were analyzed using HOMER (http://homer.ucsd.edu/homer/) to calculate the reads per kb per million mapped reads (RPKM) of RefSeq genes^{49 (link)}. Expression levels were compared with genes whose average RPKM values over 100, and GO analysis was performed using DAVID Bioinformatics Resources 6.8 and GeneSpring software (Agilent Technologies, Palo Alto, CA, USA).

Following FACS sorting, the viability and purity of isolated neutrophils was tested immediately once the cell sorting was finished. Only the sorted samples with over 98% DAPI negative (living cells) and over 90% DsRed positive (neutrophil marker) were processed for the RNA extraction using the RNeasy Micro Kit (Qiagen, 74004). The quality and quantity of isolated RNA were examined with Bioanalyzer by using Agilent RNA 6000 Pico Kit (Agilent Technologies, 5067-1513) and all RNA samples in this study had RNA integrity values above 7. These RNAs were processed to NGS (next generation sequencing)-qualified cDNA by using SMARTer Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories, Inc., 634848). The cDNAs were examined with Bioanalyzer by using Agilent High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). DNA shearing was performed on Covaris AFA system and the resulting DNA was in the 200–500 bp range. The sheared cDNA was prepared for constructing multiplex sequencing libraries using NEB DNA Library Prep Kits (New England Biolabs Inc., E7370L and E7335S). Each multiplex occupied one lane and was sequenced on Illumina HiSeq NGS platform with the sequencing read length of 100 bp.