title: RNA-seq of wt mus macrophages  infected with H37Rv and PTK2B siRNA
design: RNA library construction Experimental steps: 1. mRNA isolation and fragmentation a. take 500ng total RNA, adjust the volume to 50ul with water, add 50ul mRNA capture beads and mix well; b. place it on the PCR instrument at 65  for 5min, and then place it at room temperature for 5min; c. put it on the magnetic rack for 5min and remove the supernatant; d. remove it from the magnetic frame, add 200ul wash buffer and mix well; e. put it on the magnetic rack for 5min and remove the supernatant; f. remove it from the magnetic frame, add 50ul trisbuffer and mix well; g. place it on the PCR instrument at 80 degrees for 2min and 25 degrees hold; h. add 50ul beadbinding buffer, mix well, and place at room temperature for 5min; i. put it on the magnetic rack for 5min and remove the supernatant;t j. remove it from the magnetic frame, add 200ul wash buffer and mix well; k. put it on the magnetic rack for 5min and remove the supernatant; l. add 19ul frame / prime buffer and mix well; m. put it on the PCR instrument at 94 degrees for 3min and 4 degrees for hold; n. put it on the magnetic rack for 5min, take out 16ul to a new EP tube, and prepare 0.12mg/ml actinomysin D solution; 2.Double stranded cDNA a. add 26ul of 1st strand buffer, 22ul of 1st strand enzyme mix and 1ul of actinomycin D (0.12 mg / ml) into 16ul supernatant and mix well; b. put it on the PCR instrument, 25 degrees for 10min, 42 degrees for 15min and 70 degrees for 15min; c. add 25ul of 2nd strand buffer 2 (with dUTP) and 15ul of 2nd strand enzyme super mix into the reaction product of the previous step, and mix well; d. put it on the PCR instrument, 16 degrees for 30min and 65 degrees for 15min; 2. Add a connector a. add 25ul rapid ligation buffer3, 5ul rapid DNA ligase2, 2 UL connector, 3ul water, mix well, place it on the PCR instrument for 20 degrees and 15min; b. add 45ul DNA clean beans, mix well, and place at room temperature for 10min; c. place the magnetic rack at room temperature for 5min, remove the supernatant and do not move the beads; d. add 200 UL of 80% ethanol for 30 s at room temperature, remove the supernatant and repeat once; e. dry at room temperature for 10min; f. add 53ul water, remove it from the magnetic frame, fully mix it, and place it at room temperature for 5min; g. place it on the magnetic frame and take the supernatant 50ul into a new pipe. h. add 35ul DNA clean beans, mix well, and place at room temperature for 10min; i. place the magnetic rack at room temperature for 5min, remove the supernatant and do not move the beads; j. add 200 UL of 80% ethanol for 30 s at room temperature, remove the supernatant and repeat once; k. Dry at room temperature for 10min; l. add 22ul water, remove it from the magnetic frame, fully mix it, and place it at room temperature for 5min; m. place it on the magnetic frame and take the supernatant 19ul into a new pipe.  4. PCR enrichment a. add 19uldna, 2.5ul i7 primer+2.5ul i5 primer, 25ulhifi amplification mix and 1ulheat labile UDG successively; b. 37  10min, 98  30s, 15 cycles (98  10s, 60  30s, 72  30s), 72  5min, 4  hold; c. add 45ul amurebeads, mix well, and place at room temperature for 15min; d. place the magnetic rack at room temperature for 10min, remove the supernatant and do not move the beads; e. add 200 UL of 80% ethanol for 30 s at room temperature, remove the supernatant and repeat once, f. dry at room temperature for 10min; g. add 33ul water, remove it from the magnetic frame, fully mix it, and place it at room temperature for 5min; h. place it on the magnetic frame and take the supernatant 30ul into a new pipe.