GSM5525999: pmIA.Hic1_2; Mus musculus; RNA-Seq
Single-cell suspensions of splenocytes were prepared by mechanical disaggregation followed by treatment with ACK lysis buffer. 5e3 empty vector transduced and 5e3 Hic1-Overexpressing P14 cells were sorted into PBS + 5% BSA. To improve sample purity, 1e3 cells were then resorted into 1x TCL lysis buffer + 1% 2-mercaptoethanol. Samples were prepared using Immgen's standard protocol for ultra-low input RNA-seq (https://www.immgen.org/img/Protocols/ImmGenULI_RNAseq_methods.pdf). Excerpt here: 5 ul of the lysates are used for each sample for Smart-seq2 library construction, prepared as  previously described (Picelli et al., 2013; Picelli et al., 2014) with slight modifications. Briefly, total RNA was  captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected  using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) and converted to  cDNA via reverse transcription. First strand cDNA was subjected to limited PCR amplification followed by Tn5  transposon based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were  then PCR amplified for 12 cycles using barcoded primers such that each sample carries a specific  combination of eight base Illumina P5 and P7 barcodes and pooled together prior to sequencing.