GSM4726557: cd69_minus_wt_2; Mus musculus; RNA-Seq
For RNA sequencing, TCRb-low CD69-CD44- and TCRb-low CD69+CD44- DP thymocytes from control Lsd1fl/fl and CD2-iCre;Lsd1fl/fl mice were sorted using a FACSAria (BD Bioscience). Four biological replicates for each group. Total RNA was extracted using Arcturus PicoPure RNA Isolation Kit (Cat#: 12204-01, Applied Biosystems by Thermo Fisher Scientific). The libraries were sequenced using HiSeq Rapid run with 2 x 100bp sequencing. For ChIP-Seq, whole thymi were dissected, processed and thymocytes were fixed with 1% formaldehyde. Fixation was quenched with 0.125M glycine. Thymocytes were lysed using a lysis buffer containing: 5 mM PIPES (pH8), 85 mM KCl, 10ul/mL Ipegal and protease inhibitor. Lysates were then homogenized, and the nuclei lysed using a nuclear lysis buffer containing: 10mM Tris HCl (pH8), 10 mM EDTA (pH8), 1% SDS (v/v), 10 ul/ml protease inhibitor. Isolated chromatin was sonicated to 200-500 bp fragments (Diagenode Bioruptor). Sonicated DNA was then incubated with Protein A beads (Invitrogen Dynabeads) conjugated to H3K4Me3 (Milipore, MC315), H3K4Me2 (Milipore, AW30), or H3K4Me1 (abcam, ab8895) antibodies.  Immunoprecipitated DNA fragments were isolated using a Takara DNA isolation kit. RNA-Seq libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Cat#: RS-122-2101, Illumina) with PolyA purification. Each sample use 1ug total RNA-Seq input. For ChIP-Seq, DNA libraries were prepared using a DNA SMART CHIP Seq Kit (Takara) RNA-Seq: 2 x 101 bp paired-end sequencing, ChIP-Seq: 1x51 bp single-end sequencing.