title: GSM4762441: TAM062119_005; Mus musculus; RNA-Seq
library_construction_protocol: HSPCs (~12,000 cell/LSK population) from P15 wild-type (n=4) or Gabbr1 null (n=4) BM were sorted (MoFlo Astrios) and collected in Trizol LS (Invitrogen). Volumes were adjusted to a final ratio of 3:1 for Trizol LS and nuclease-free water. Samples were stored at -80°C until total RNA was isolated. Then samples were thawed, equilibrated to room temperature, and divided into 800 μl aliquots. 5 μg of Sigma “GenElute” linear polyacrylamide (LPA; Cat. No. 56575-1ML), diluted in nuclease-free water, was added to each aliquot and vortexed for 30 seconds. Chloroform extraction and 3 ethanol washes were performed as per the Trizol product insert (Invitrogen protocol Cat. No. 10296-010). The pellet was resuspended in 6 μl RNAse-free water. RNA samples were quantified and purity and integrity were checked using NanoDrop™ One Spectrophotometer (Thermo Scientific) and Agilent 4200 TapeStation. NGS library preparation was completed using NuGen's Ovation® SoLo RNA-Seq Systems, per their User Guide M01406 v4, including a targeted depletion with SoLo AnyDeplete Mouse Probe Mix. RNA input into library preparation was between 3.6 - 5 ng. The number of cycles for the first library PCR amplification were optimized using qPCR, as outlined in the SoLo User Guide, with the Prime Pro 48 Real-Time PCR system (Techne). Completed libraries were quantified using Qubit fluorometer (Invitrogen) and size was determined using TapeStation. Libraries were pooled based on Qubit dsDNA concentration and run on MiSeq for index balancing. The second pool, with corrected library inputs based on the %Reads Identified (PF) results from the MiSeq run, was purified with 1:1 Agencourt AMPure XP beads. The final, purified pool was quantified using Qubit and pool size was determined using TapeStation. Sequencing was run on a NovaSeq6000 SP flow cell, 2x50 bp, two lanes, at the University of Illinois Roy J. Carver Biotechnology Center High-Throughput Sequencing and Genotyping Unit.