title: GSM9252549: CD4+T cells，KO-4; Mus musculus; RNA-Seq
library_construction_protocol: The RNA of the total sample was separated and purified using TRIzol (thermofisher, 15596018) in accordance with the operation plan provided by the manufacturer. Then the quantity and purity of total RNA were controlled by NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and the integrity of RNA was detected by Bioanalyzer 2100 (Agilent, CA, USA). The concentration >50ng/μL, the RIN value >7.0, and the total RNA>1μg meet the requirements for downstream experiments. The mRNA containing PolyA (polyadenylic acid) was specifically captured by two rounds of purification using oligo(dT) magnetic beads (Dynabeads Oligo (dT), cat.25-61005, Thermo Fisher, USA). The captured mRNA was fragmented under high-temperature conditions using the Magnesium RNA Fragmentation Module (NEBNextR magnesium RNA Fragmentation Module, cat.E6150S, USA) at 94 ° C for 5-7 minutes. cDNA was synthesized from fragmented RNA under the action of Invitrogen SuperScriptTM II Reverse Transcriptase (cat.1896649, CA, USA). Then, double-stranded synthesis was carried out using E. coli DNA polymerase I (NEB, cat.m0209, USA) and RNase H (NEB, cat.m0297, USA) to convert these complex double-stranded DNA and RNA into DNA double-stranded. Meanwhile, dUTP Solution (Thermo Fisher, cat.R0133, CA, USA) was incorporated into the two strands to complete the ends of the double-stranded DNA into flat ends, and then an A base was added to each end to enable it to connect with the connector with a T base at the end. And the size of the fragments was screened and purified by using magnetic beads. The two strands were digested with UDG enzyme (NEB, cat.m0280, MA, US), then subjected to PCR - pre-denaturation at 95 ° C for 3 minutes, denaturation at 98 ° C for a total of 8 cycles, each lasting 15 seconds, annealing to 60 ° C for 15 seconds, extension at 72 ° C for 30 seconds, and finally extension at 72 ° C for 5 minutes. Form a library (chain-specific library) with a fragment size of 300bp±50bp. Finally, we performed double-ended sequencing on it using illumina NovaseqTM 6000 (LC Bio Technology CO.,Ltd. Hangzhou, China) in accordance with standard operation, with the sequencing mode being PE150.