NextSeq 500 sequencing; RNA-seq of mouse wildtype HSCs purified from recipient mice co-transplanted with wildtype or different types of lymphoid cell deficient HSCs
RNA-seq of mouse wildtype HSCs purified from recipient mice co-transplanted with wildtype or different types of lymphoid cell deficient HSCs
Wildtype (WT), B6.129S2(B6)-Ighmtm1Cgn/J (uMT-/-), or NOD-scid IL2Rgamma null (NSG) HSCs (lineage (CD3, CD4, CD8, B220, Gr1, Mac1, Ter119)-/ckit+/Sca1+/Flk2-/CD34-/CD150+) were obtained from the crushed bones of donor mice and isolated using FACS sorting with the FACS-Aria II (BD Biosciences, San Jose, CA) after enrichment using CD117 microbeads (AutoMACS, Miltenyi Biotec, Auburn, CA). WT, uMT-/-, and NSG HSCs were isolated as described above. In addition to the donor HSCs, we transplanted each recipient mouse 250,000 whole bone marrow cells (helper cells) flushed from the femurs of F1 mice (CD45.1/ CD45.2) mice. These helper cells contain HSCs and other hematopoietic progenitors that assist in blood production. HSCs derived from NSG mice lack the ability to respond to Il-7 and hence cannot produce lymphoid cell lineages. We transplanted 1,500 WT HSCs and 3,000 NSG HSCs into each recipient, and we found the expected 1:2 ratio in donor-derived granulocytes. All donor and recipient mice were 2-3 months old at the time of transplantation. Irradiation was performed on all recipient mice before transplantation at 950 cGy. We examined 3 mice for each experimental group, and performed biological replicates shown in appendix figures. Mice were bred and maintained at the Research Animal Facility of the University of Southern California. Animal procedures were approved by the Institutional Animal Care and Use Committee. Recipient mice were 9-11 months old when WT HSCs were purified from their bone marrow for RNA sequencing analysis. RNA was isolated from 4,000 HSCs  per mouse using the Zymo Research (Irvine, CA) Quick-RNA MicroPrep RNA was isolated from 4,000 HSCs (lineage (CD3, CD4, CD8, B220, Gr1, Mac1, Ter119)-/ckit+/Sca1+/Flk2-/CD34-/CD150+). Starting material was (varied by experiment, was anywhere from 50 ng to 100 ng) of total RNA. Prior to library construction RNA integrity was verified by Experion analysis (Bio-Rad). For B6.129S2(B6)-Ighmtm1Cgn/J (uMT-/-) mice and the related control mice, polyA selection was carried out using Illumina Truseq V2 polyA beads. Material enriched for polyA was then introduced into SMART-Seq v4 Ultra Low Input RNA Kit (TaKaRa) at the elute-fragmentation-priming step then taken through the protocol according to manufacturer's instructions. For NOD-scid IL2Rgamma null (NSG) mice and the related control mice, total RNA was fragmented first and then processed according to manual instruction using SMARTer® Stranded Total RNA-Seq Kit v2 (TakaRa). Final PCR cycle numbers vary according to input amounts, for this set (low input got 12-14 cycles, higher input amounts 6-10) cycles was performed.  Libraries were visualized by Bioanalyzer analysis (Agilent) and quantified by qPCR (Kapa Biosystems Illumina Library Quantification Kit).