TITLE: MAPseq (Multiplexed Analysis of Projections by Sequencing) sample processing protocol
AUTHORS: Huiqing Zhan, Justus Kebschull, Anthony M. Zador

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes sample processing steps of MAPseq, a high-throughput mapping of single-neuron projections by sequencing of barcoded RNA, as described in details by Kebschull et al.., 2016 . In MAPseq, a brain area of interest is infected with a Sindbis barcoded library. After 40-44 hrs, the injection and the projection sites of interest are dissected, processed and sequenced using this protocol.  </div></div>

[STEPS]
?. [RNA extraction]
Tissues are homogenized in 400ul of Trizol either with a Pellet Pestle Motor or with a tissue homogenizer, and then add 600ul of Trizol to make 1 ml of Trizol/sampleExtract RNA according to manufacturer’s protocol of Trizol ReagentDissolve RNA of each tissue sample into 13 ul of H2O
?. [Reverse transcription with Superscript IV ]
One reaction per sample.For each reaction, add the following reagents(total of 13ul) into each well:4 ul of RNA from each sample6 ul of H2O 1 ul of spike-in RNA (Spike-in RNA has its unique barcode and is used to normalize for varying reverse transcription, PCR or library making efficiencies)1ul of 10uM RT primer (each RT primer contains a random 12-nt unique molecular identifier (UMI) to tag each individual barcode mRNA molecule and a 8-nt slice specific identifier (SSI) to tag each sample)1 ul of 10mM dNTPMix well.
?. [RNA extraction]
Each fresh frozen brain area of interest is dissected and kept in RNAse-free tubes at -80°C before RNA extraction.
?. [Reverse transcription with Superscript IV ]
Incubate at 70 °C for 10 minutes, then immediately transfer samples to ice.Incubate on ice for 5 minutes.
?. [Reverse transcription with Superscript IV ]
Add per well (total of 7ul)4ul SSIV buffer 1ul 0.1M DTT 1ul RNasin1ul SSIVMix well.
?. [Reverse transcription with Superscript IV ]
Incubate the mixture in a thermocycler10min at 55 °C10min at 80 °CStore samples at 4 °C or -20ºC till next step.
?. [AMPure XP beads clean up]
Pool all of the RT product from targets or injection sites. Do not mix target site with injection site.Mix well.
?. [AMPure XP beads clean up]
For pooled samples from target sitesAdd 1.8X AMPure XP beadsMix by pipetting 10 timesIncubate for 10 minutes at room temperatureAliquot into several tubes for easier elutionPut on magnetic rack, wait for 2-3 minutes till solution is clearOn magnetic rack, discard supernatant Wash twice with fresh 80% EtOH to cover the pellet for 30 secondsAir dry pellet till  Resuspend beads in (17 * (total number of target site/10) ul) of H20Incubate at room temperature for 5 minutesOn magnetic rack, collect all of supernatant in an Eppendorf tube.
?. [AMPure XP beads clean up]
For pooled samples from injection sitesAdd 1.8X AMPure XP beadsMix by pipetting 10 timesIncubate for 10 minutes at room temperatureAliquot into several tubes for easier elutionPut on magnetic rack, wait for 2-3 minutes till solution is clearOn magnetic rack, discard supernatant Wash twice with fresh 80% EtOH to cover the pellet for 30 secondsAir dry pellet  Resuspend beads in (17 * (total number of target site/5) ul) of H20Incubate at room temperature for 5 minutes On magnetic rack, collect all of supernatant in an Eppendorf tube.
?. [2nd strand cDNA synthesis]
Per 17ul of bead purified product, add2.4ul SSIV buffer 0.6ul 0.1M DTT5.6ul Second Strand buffer 0.75ul 10mM dNTPs0.25ul E coli DNA ligase 1ul DNA polymerase I 0.25ul RNaseH Mix well.
?. [2nd strand cDNA synthesis]
Incubate the mixture in a thermocycler 2 hours at 16ºCAdd 1ul of T4 DNA polymerase Incubate for 10 minutes at 16ºC.Store sample at 4ºC or -20ºC till next step.
?. [Beads clean up]
Pool all 2nd stranding reactions, measure the total volumeAdd 1.8x AMPure XP beadsMix by pipetting 10 timesIncubate for 10 minutes at room temperaturePut on magnetic rack, wait for 2-3 minutes till solution is clearOn magnetic rack, discard supernatant Wash twice with fresh 80% EtOH to cover the pellet for 30 secondsAir dry pellet  Resuspend beads in (16 * (total number of 2nd strand reaction) ul) of H20Incubate at room temperature for 5 minutes On magnetic rack, collect all of supernatant in a PCR tube.
?. [Exonuclease treatment]
Per 16ul of beads purified product, add2ul Exonuclease buffer 2ul Exo IMix well.
?. [Exonuclease treatment]
Incubate the mixture in a thermocycler1 hr at 37 ºCHeat inactivate at 80 ºC for 20 minutesStore sample at 4ºC or -20ºC till next step.
?. [PCR I]
Per 20ul Exonuclease reaction, add25ul Accuprime buffer25ul 10uM nested1st gfpF primer25ul 10uM nested2nd primer2.5 ul Accuprime Pfx HF enzyme152.5ul H20Mix well and aliquot the mixture into PCR tubes
?. [PCR I]
Perform thermal cycling as follows:Initial denaturation at95ºC 2 minutesRun 15 cycles of 95ºC 15 seconds68ºC 2.5 minutesFinal extension at68ºC 5 minutes
?. [Exonuclease treatment]
Add 5 ul of Exo I to each 50 ul PCR reactionIncubation at 37ºC for 30 minutesHeat inactivation at 80ºC for 20 minutes
?. [Test PCR II]
Test optimal PCR II cycle number in a 25ul reaction/test by adding2.5ul of 1/10 diluted PCR I product2.5ul Accuprime buffer2.5ul 10uM Sol I primer2.5ul 10uM Sol II primer0.25ul Accuprime Pfx HF enzyme14.75ul H20
?. [Test PCR II]
For pooled target sites, run 14, 17, 20, 23, 26 and 29 cyclesFor pooled injection sites, run 11, 14, 17, 20, and 23 cyclesPerform thermal cycling as follows:Initial denaturation at95ºC 2 minutesRun different cycles of 95ºC 15 seconds68ºC 1 minutesFinal extension at68ºC 5 minutes
?. [Test PCR II]
Load test PCR II products on 2% agarose gel to find out the lowest cycle number which could generate a single and clean 230bp band.
?. [PCR II]
For pooled injection sites, use 12 ml of PCR II reactionFor pooled target sites, use 3 ml of PCR II reaction for 40 samples.
?. [PCR II]
Perform thermal cycling as follows:Initial denaturation at95ºC 2 minutesRun optimal cycles of 95ºC 15 seconds68ºC 2.5 minutesFinal extension at68ºC 5 minutes
?. [PCR II]
In each PCR II reaction, Add the same concentration of PCR I product as what is used in the test PCR II1x Accuprime buffer1uM Sol I primer1uM Sol II primer1U of Accuprime Pfx HF enzyme/50ul of reaction
?. [Gel purification]
Purify PCR product using Wizard® SV Gel and PCR Clean-Up System from Promega and elute the product into 40ul of H2O per 1ml of PCR product
?. [Gel purification]
Load purified PCR product into 2% agarose gel
?. [Gel purification]
Cut ~230bp band from the gel and purify the product with MinElute Gel Extraction Kit from Qiagen
?. [Bioanalyzer test with Agilent High Sensitivity DNA Kit]
Load purified 230bp PCR product on a DNA bioanalyzer chip using Agilent High Sensitivity DNA Kit to confirm its size and quantity before submitting for sequencing
?. [Next Generation Sequencing (NGS)]
Submit purified 230bp product for an Illumina NextSeq500 high output run at paired end 36 using the SBS3T sequencing primer for paired end 1 and the Illumina small RNA sequencing primer 2 for paired end 2.