title: GSM8547705: N093_Control_Stroma; Mus musculus; RNA-Seq
library_name: GSM8547705
library_construction_protocol: RNA was isolated using guanidinium thiocyanate-phenol-chloroform extraction (TRIzol®) from sorted BM samples. Due to the low numbers of cells isolated from the sorted bone marrow stroma RNA, samples was concentrated using SPRIselect magnetic beads (Beckman Coulter). For the hematopoietic samples: Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina NovaSeq6000 at PE150. Each library was sequenced to 6G (20 million paired-end reads).  For the Stromal Samples: Libraries were created using the NEBNext Low Input RNA Library Prep Kit (E6420S, New England Biolabs) and indexed using the NEBNext Multiplex Oligos dual index primers (E6440S, New England Biolabs). Libraries were sequenced at Novogene (Sacramento, CA) using the NovaSeq X system to generate 150bp paired end reads.